Compositions and methods for the delivery of nucleic acids

ABSTRACT

A nanosized complex includes a nucleic acid and a compound comprising formula (I): 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  is an alkylamino group or a group containing at least one aromatic group; 
             R 2  and R 3  are independently an aliphatic group or hydrophobic group; 
             R 4  and R 5  are independently H, a substituted or unsubstituted alkyl group, an alkenyl group, an acyl group, or an aromatic group, or a polymer, a targeting group, a detectable moiety, or a linker, or a combination thereof, and at least one of R 4  and R 5  includes a retinoid or retinoid derivative that targets and/or binds to an interphotoreceptor retinoid binding protein; 
             a, b, c, and d are independently an integer from 1 to 10; and pharmaceutically acceptable salts thereof.

RELATED APPLICATION

This application claims priority to U.S. Provisional Ser. No.62/880,327, filed Jul. 30, 2019, this application is also aContinuation-in-Part of U.S. Ser. No. 15/767,119, filed Apr. 9, 2018,which is a National Phase of PCT/US2016/056453, filed Oct. 11, 2016,which claims priority to U.S. Provisional Application No. 62/239,306,filed Oct. 9, 2015, the subject matter of which is incorporated hereinby reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. EB00489awarded by The National Institutes of Health and Grant NumberDGE-0951783, awarded by the National Science Foundation GraduateResearch Fellowship. The United States government has certain rights tothe invention.

BACKGROUND

The eye is an attractive target for gene therapy strategies due to itsaccessibility and immune-privileged characteristics, which will minimizethe inflammatory and immune reactions when local gene therapy isapplied. However, gene delivery to the eye is never an easy task,because there are multiple obstacles in the way of genetic therapeuticsto their destination. Retina is separated into ten layers and each ofthem has vital functions in the process of photoreception/transductionprocesses. Genetic eye disorders in each specific layer can causedifferent phenotypes. Therefore, gene therapies for these disordersrequire not only high transfection efficiency but also high specificity,because therapies without high specificity will introduce exogenousfunctional gene to undesired tissue, which will in turn result inpotential disorders of vision.

Non-viral systems that employ cationic lipids, dendrimers, polycationsand polysaccharides have been developed for gene delivery. Non-viralsystems generally exhibit advantages of the ease of production, goodsafety profiles, and unlimited cargo capacity. However, their clinicaltranslation is hindered by their low transfection efficiency andtransient gene expression. Novel design of highly effective non-viraldelivery systems is needed to overcome the limitations of the existingnon-viral delivery systems for effective gene therapy of visualdisorders.

SUMMARY

Embodiments described herein relate to compounds used to formmultifunctional pH-sensitive carriers or nanoparticles that are designedto condense therapeutic nucleic acids and deliver the condensed nucleicacids to cells of the eye. The compounds can include a protonable aminohead group, which can complex with the nucleic acids, fatty acid orlipid tails, which can participate in hydrophobic condensation, twocysteine residues capable of forming disulfide bridges viaautooxidation, and a targeting group that targets and/or binds to aretinal or visual protein, such as an interphotoreceptor retinoidbinding protein.

In some embodiments, the compound includes formula (I):

-   -   wherein R¹ is an alkylamino group or a group containing at least        one aromatic group; R² and R³ are independently an aliphatic        group or a hydrophobic group derived, for example, from a fatty        acid; R⁴ and R⁵ are independently H, a substituted or        unsubstituted alkyl group, an alkenyl group, an acyl group, or        an aromatic group, or includes a polymer, a targeting group, or        a detectable moiety and at least one of R⁴ and R⁵ includes a        targeting group that targets and/or binds to a retinal or visual        protein, such as an interphotoreceptor retinoid binding protein;        a, b, c, and d are independently an integer from 1 to 10 (e.g.,        a, b, c, and d are each 2); and pharmaceutically acceptable        salts thereof.

In some embodiments, R¹ can include at least one of:

-   -   where R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are        independently hydrogen, an alkyl group, a hydrophobic group, or        a nitrogen containing substituent; and e, f, g, i, j, k, and l,        are an integer from 1 to 10. For example, R¹ can include at        least one of CH₂CH₂NH₂, CH₂CH₂NHCH₂CH₂NHCH₂CH₂NH, or        CH₂CH₂NHCH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NH.

In other embodiments, R² and R³ are independently a hydrophobic groupderived from oleic acid or linoleic acid and are the same or different.

In other embodiments, the targeting group that targets and/or binds toan interphotoreceptor retinoid binding protein can be covalentlyattached to a thiol group of a cysteine residue of the compound by alinker. For example, the targeting group can include a retinoid orretinoid derivative, such as all-trans-retinylamine or(1R)-3-amino-1-[3-(cyclohexylmethoxy)phenyl]propan-1-ol; hydrochloride,that is conjugated to a linker, such as a polyethylene glycol linker,which is covalently bound to the thiol group of a cysteine residue ofthe compound.

In some embodiments, a therapeutic nucleic acid can be complexed withthe compound to form multifunctional pH-sensitive carriers ornanoparticles that can be administered to the eye of a subject in needthereof. The therapeutic nucleic acid can include any nucleic acid thatwhen complexed with the compound and introduced to or within the eye iscapable of treating, ameliorating, attenuating, and/or eliminatingsymptoms of a disease or disorder of the eye.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-C) illustrate images and a graph showing in vitro transfectionof ARPE19 cells with ECO/pDNA nanoparticles. (A) Confocal microscopyimages and (B) flow cytometry analysis of ARPE19 cells transfected withECO/pGFP (N/P=6) nanoparticles and Lipofectamine 2000/pGFP nanoparticlesfor 48 hours (** p<0.005). (C) Confocal fluorescence microscopic imagesdemonstrating intracellular trafficking of ECO/Cy3-pDNA nanoparticles inARPE-19 cells. Cells were treated with LysoTracker Green (1:2500dilution) and Hoechst 33342 (1:10000 dilution) and then transfected withECO/Cy3-labeled nanoparticles at N/P=6. After 1, 4 and 24 h oftransfection, cells were fixed and imaged. Arrows denote theECO/Cy3-pDNA nanoparticles. Scale bar is 20 μm.

FIGS. 2(A-D) illustrate synthesis and MALDI-TOF mass spectrum ofRet-PEG-MAL and TEM images of Ret-PEG-ECO/pDNA nanoparticles.Nanoparticles were prepared by depositing 20 μL of the particle solutiononto a 300-mesh copper grid covered by a thin amorphous carbon film (20nm). Samples were stained twice by the addition of 3 μL of 2% uranylacetate aqueous solution.

FIGS. 3(A-B) illustrate images showing in vivo gene transfection withtargeted Ret-PEG-ECO/pGFP nanoparticles in wild type BALB/c mice. Mice(1-month-old) were subretinally injected with ECO/pGFP orRet-PEG-ECO/pGFP nanoparticles. RPE flat mounts were obtained 3 dayspost transfection. (A) Fluorescence microscopic images show enhanced GFPexpression with Ret-PEG-ECO/pGFP nanoparticles in the RPE 3-days postinjection. (B) Confocal fluorescence microscopic images show GFPexpression specifically in the RPE with anti-ZO-1 antibody staining(white). The tight junction protein ZO-1 represents the borders of theRPE cells.

FIGS. 4(A-F) illustrate graphs and a plot showing gene replacementtherapy using Ret-PEG-ECO/pRPE65 nanoparticles in rpe65^(−/−) mice. Themice were subretinally injected with Ret-PEG-ECO/pRPE65 nanoparticles orRet-PEG-ECO. (A) Relative RPE65 mRNA level in treated (pRPE65 injected)and control groups 15 days after treatment. (B) Representative scotopicand photopic electroretinograms acquired in rpe65^(−/−) mice under theintensity of 1.6 log cd×s/m² at 7 days after the treatment. Amplitudesof (C) scotopic a-waves, (D) photopic a-waves, (E) scotopic b-waves and(F) photopic b-waves of treated and control rpe65^(−/−) mice at 3, 7, 30and 120 days post injection.

FIGS. 5(A-C) illustrate images showing cone preservation after genereplacement therapy using Ret-PEG-ECO/pRPE65 nanoparticles inrpe65^(−/−) mice 120 days after treatment. Peanut agglutinin was used tostain cone photoreceptors. Nuclei were stained with DAPI.

FIGS. 6(A-D) illustrate graphs showing the therapeutic effect of genereplacement therapy using Ret-PEG-ECO/pRPE65 nanoparticles in3-month-old rpe65^(−/−) mice. ERG amplitudes of major response waveforms(A) scotopic a-waves, (B) photopic a-waves, (C) scotopic b-waves and (D)photopic b-waves in the treatment and control groups of rpe65^(−/−)mice.

FIGS. 7(A-E) illustrate plots and graphs showing safety assessment ofRet-PEG-ECO/pRPE65 nanoparticles in BALB/c mice (1-month-old). (A)Representative ERG traces of scotopic waveforms in the PEG-ECO/pRPE65treated group and untreated mice at 30 days post-injection. ERGamplitudes of amplitudes of (B) scotopic a-waves, (C) photopic a-waves,(D) scotopic b-waves, and (E) photopic b-waves in treated and controlmice.

FIGS. 8(A-B) illustrate a schematic and spectra showing design ofACU-PEG-HZ-MAL Targeting Ligand. (A) Synthetic route of ACU-PEG-HZ-MALtargeting ligand. (B) MALDI-TOF mass spectra of ACU-PEG-HZ-MAL andintermediates.

FIGS. 9(A-F) illustrates a schematic, graphs, plots, and images showingformulation and characterization of ACU4429 modified ECO/pABCA4nanoparticles. (A) Scheme of ACU-PEG-HZ-ECO/pDNA nanoparticleformulation, (B) nanoparticle sizes, (C) size distributions, (D) zetapotentials, (E) zeta potential distributions, and (F) confocal images ofGFP expression 24 h after transfection in ARPE-19 cells under 10% serumtransfection media using ECO/pCMV-GFP nanoparticles of N/P ratios 6, 8,10 and 12. (n=3, error bars=±SD, **P<0.05. Scale bars represent 20 μm.).

FIGS. 10(A-B) illustrate an image and graph showing stability,encapsulation and cytotoxicity of ECO/pABCA4 and ACU4429 modifiedECO/pABCA4 nanoparticles. (A) Agarose gel electrophoresis of ECO/pABCA4,PEG-ECO/pABCA4 and ACU-PEG-HZ-ECO/pABCA4 nanoparticles with PEG ligandsas controls (The arrow indicated the DNA smear of free pABCA4 notencapsulated by short pipette mixing of ECO and pABCA4.). (B) Viabilityof ARPE-19 cells treated with ECO/pABCA4 and ACU4429 modified ECO/pABCA4nanoparticles, and free ECO, PEG ligands and non-treated ARPE cells ascontrol (significance analysis was done using one-way ANOVA, p<0.05).

FIGS. 11(A-C) illustrate images and graphs showing in vitro transfectionefficiency of ACU-4429-PEG-HZ-ECO/pDNA nanoparticles. (A) Confocalfluorescence images (B) Flow cytometry measurements of the cytosolicdelivery of ACU-PEG-HZ-ECO/Cy3-pDNA and PEG-ECO/Cy3-pDNA nanoparticlesat an N/P ratio of 10 in ARPE-19 cells. DNA plasmid was labeled withCy3, nuclei with Hoechst 33342, and late endosomes with LysoTrackerGreen. Flow cytometry recorded mean fluorescence intensities of Cy3positive ARPE-19 cells at each time point. (C) qRT-PCR of ABCA4 mRNAexpression at 48 h in ARPE-19 cells transfected withACU-PEG-HZ-ECO/pCMV-ABCA4 and PEG-ECO/pCMV-ABCA4 nanoparticles (n=3,error bars=±SD, **, ^(##P)<0.05. Scale bars represent 20 μm).

FIGS. 12(A-B) illustrate images and graphs showing in vivo evaluation ofthe targeted ACU-PEG-HZ-ECO/pDNA nanoparticles for gene delivery to theRPE. (A) The distribution of ECO/Cy3-pDNA nanoparticles andACU-PEG-HZ-ECO/Cy3-pDNA nanoparticles in the subretinal space ofAbca4^(−/−) mice 4 days after subretinal injection (Control: PBSinjection; IPM: interphotoreceptor matrix; ONL: outer nuclear layer;INL: inner nuclear layer). (B) qRT-PCR analysis of ABCA4 mRNA expressionin Abca4^(−/−) mice 7 days after subretinal injection of ECO/pCMV-ABCA4and ECO/pRHO-ABCA4 nanoparticles modified with PEG and ACU-PEG-HZ (n=3,error bars=±SD, **P<0.05. Scale bars represent 50 μm).

FIG. 13 illustrates a schematic showing the targeting mechanism ofACU-PEG-HZ-ECO/pDNA nanoparticles for gene delivery in the RPE. Onceinjected into the subretinal space, ACU-PEG-HZ-ECO/pDNA nanoparticlesbind to interphotoreceptor retinoid binding protein (IRBP) in theinterphotoreceptor matrix. IRBP binding helps transport thenanoparticles to the targeted RPE cells. Following endocytosis, the PEGlayer of the targeted nanoparticles sheds off due to the acid-catalyzedhydrolysis of the hydrazone linker in the acidic endosome. The ECO/pDNAnanoparticles then escape from the endosomal entrapment and then releasethe therapeutic plasmid DNA through the PERC mechanism.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York,1991, and Lewin, Genes V, Oxford University Press: New York, 1994. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of thepresent invention.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like. “Optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where the event or circumstanceoccurs and instances where it does not. For example, the phrase“optionally substituted lower alkyl” means that the lower alkyl groupcan or cannot be substituted and that the description includes bothunsubstituted lower alkyl and lower alkyl where there is substitution.

The term “alkenyl group” is defined herein as a C₂-C₂₀ alkyl grouppossessing at least one C═C double bond.

The term “alkyl group” as used herein is a branched or unbranchedsaturated hydrocarbon group of 1 to 25 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and thelike. A “lower alkyl” group is an alkyl group containing from one to sixcarbon atoms.

The term “acyl” group as used herein is represented by the formulaC(O)R, where R is an organic group such as, for example, an alkyl oraromatic group as defined herein.

The term “alkylene group” as used herein is a group having two or moreCH₂ groups linked to one another. The alkylene group can be representedby the formula (CH₂)_(a), where a is an integer of from 2 to 25.

The term “aromatic group” as used herein is any group containing anaromatic group including, but not limited to, benzene, naphthalene, etc.The term “aromatic” also includes “heteroaryl group,” which is definedas an aromatic group that has at least one heteroatom incorporatedwithin the ring of the aromatic group. Examples of heteroatoms include,but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Thearyl group can be substituted or unsubstituted. The aryl group can besubstituted with one or more groups including, but not limited to,alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone,aldehyde, hydroxy, carboxylic acid, or alkoxy.

The phrase “nitrogen containing substituent” is defined herein as anyamino group. The term “amino group” is defined herein as a primary,secondary, or tertiary amino group. In the alternative, the nitrogencontaining substituent can be a quaternary ammonium group. The nitrogencontaining substituent can be an aromatic or cycloaliphatic group, wherethe nitrogen atom is either part of the ring or directly or indirectlyattached by one or more atoms (i.e., pendant) to the ring. The nitrogencontaining substituent can be an alkylamino group having the formulaRNH₂, where R is a branched or straight alkyl group, and the amino groupcan be substituted or unsubstituted.

The term “nucleic acid” refers to oligonucleotides, nucleotides,polynucleotides, or to a fragment of any of these, to DNA or RNA (e.g.,mRNA, rRNA, tRNA, miRNA, siRNA) of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent a sense orantisense strand, to peptide nucleic acids, or to any DNA-like orRNA-like material, natural or synthetic in origin, including, e.g.,iRNA, ribonucleoproteins (e.g., iRNPs). The term can also encompassnucleic acids containing known analogues of natural nucleotides, as wellas nucleic acid-like structures with synthetic backbones.

The term “subject” can refer to any animal, including, but not limitedto, humans and non-human animals (e.g., rodents, arthropods, insects,fish (e.g., zebrafish)), non-human primates, ovines, bovines, ruminants,lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc.),which is to be the recipient of a particular treatment. Typically, theterms “patient” and “subject” are used interchangeably herein inreference to a human subject.

The terms “inhibit,” “silencing,” and “attenuating” can refer to ameasurable reduction in expression of a target mRNA (or thecorresponding polypeptide or protein) as compared with the expression ofthe target mRNA (or the corresponding polypeptide or protein) in theabsence of an interfering RNA molecule of the present invention. Thereduction in expression of the target mRNA (or the correspondingpolypeptide or protein) is commonly referred to as “knock-down” and isreported relative to levels present following administration orexpression of a non-targeting control RNA.

Embodiments described herein relate to compounds used to formmultifunctional pH-sensitive carriers or nanoparticles that are designedto condense therapeutic nucleic acids and deliver the condensed nucleicacids to cells of the eye. The compounds can include a protonable aminohead group, which can complex with the nucleic acids, fatty acid orlipid tails, which can participate in hydrophobic condensation, twocysteine residues capable of forming disulfide bridges viaautooxidation, and a targeting group that targets and/or binds to aretinal or visual protein, such as an interphotoreceptor retinoidbinding protein.

The protonable amino head group can complex with therapeutic nucleicacids to form multifunctional pH-sensitive carriers or nanoparticles fordelivery of nucleic acids to cells of the eye. The amines in the headgroups contribute to the essential pH-sensitive characteristic of thecarrier system, which is important for improving endosomal escape of thenucleic acid. Greater protonation of the amino head groups can occur inthe relatively acidic environment (pH=5-6) of the endosome and lysosomecompartments after cellular uptake. This enhances electrostaticinteractions between the cationic carriers and the anionic phospholipidsof endosomal/lysosomal membranes, promoting the bilayer destabilizationand nanoparticle charge neutralization events required for efficientcytosolic release of their nucleic acid payload. By affecting the numberof amines, and thus overall pKa, of the cationic carrier, the choice ofhead group can ultimately determine the degree to which such protonationcan occur. The pH-sensitive property of the carrier system is essentialso that the nanoparticles do not affect the integrity of the outer cellmembrane and cause cell death, but instead are able to selectively fusewith and destabilize the endosomal and lysosomal membranes.

The cysteine residues can form disulfide bridges via autooxidation andreact with functional groups of other compounds, such as thosecontaining thiol groups. Once the nucleic acid is complexed with thecompound to form the multifunctional pH-sensitive carriers ornanoparticles, the thiol groups can produce disulfide (S—S) bonds orbridges by autooxidation to form oligomers and polymers orcross-linking. The disulfide bonds can stabilize the nanoparticles ofthe nucleic acid and compound and help achieve release of the nucleicacid once the nanoparticle is in the cell.

For example, when the nucleic acid is siRNA, the cleavage of disulfidebonds in the siRNA delivery systems in reductive cytoplasm canfacilitate cytoplasm-specific release of siRNA. The multifunctionalpH-sensitive carriers or nanoparticles comprising the compounds arestable in the plasma at very low free thiol concentration (e.g., 15 μM).When the compounds are incorporated into target cells, the highconcentration of thiols present in the cell (e.g., cytoplasm) willreduce the disulfide bonds to facilitate the dissociation and release ofthe nucleic acid.

The disulfide bonds can be readily produced by reacting the same ordifferent compounds before complex with the nucleic acids or during thecomplex in the presence of an oxidant. The oxidant can be air, oxygen orother chemical oxidants. Depending upon the dithiol compound selectedand oxidative conditions, the degree of disulfide formation can vary infree polymers or in complexes with nucleic acids. Thus, the compoundsincluding two cysteine residues are monomers, and the monomers can bedimerized, oligomerized, or polymerized depending upon the reactionconditions.

The fatty acid or lipid tails groups can participate in hydrophobiccondensation and help form compact, stable nanoparticles with thenucleic acids and introduce amphiphilic properties to facilitate pHsensitive escape of nanoparticles from endosomal and lysosomalcompartments. This is particularly useful when the compounds are used asin vivo delivery devices.

In general, the transfection efficiency of carriers has been shown todecrease with increasing alkyl chain length and saturation of the lipidtail groups. When saturated, shorter aliphatic chains (C12 and C14)favor higher rates of inter-membrane lipid mixing and reportedly allowfor better transfection efficiencies in vitro, as compared to in vivo,whereas the opposite is true for longer chains (C16 and C18). Typically,saturated fatty acids greater than 14 carbons in length are notfavorable for nucleic acid transfections due to their elevated phasetransition temperature and overall less fluidity than those that areunsaturated. However, it has been discovered that there exists a limit,at which point an increase in unsaturation and lipid fluidity isinversely correlated to transfection efficiency, primarily because somedegree of rigidity is required for particle stability, as evidenced bythe widespread use of cholesterol in lipid nanoparticle formulations.

In some embodiments, the targeting group that targets and/or binds to aretinal or visual protein, such as an interphotoreceptor retinoidbinding protein, can be attached to a cysteine residue of the compoundby, for example, a thiol group of the cysteine residue. The targetinggroup can include, for example, a retinoid, such as a retinylamine(e.g., all-trans-retinylamine) or retinoid derivative, such as(1R)-3-amino-1-[3-(cyclohexylmethoxy)phenyl]propan-1-ol; hydrochloride.In some embodiments, the targeting group is all-trans-retinylamine. Inother embodiments, the targeting group is(1R)-3-amino-1-[3-(cyclohexylmethoxy)phenyl]propan-1-ol; hydrochloride.In still other embodiments, the targeting group is a synthetic retinoidderivative, such as a synthetic retinoid derivative described in U.S.Pat. Nos. 7,951,841 or 7,982,071 and PCT/US2015/062343 all of which areincorporated by reference in their entirety.

For example, the targeting group can include a primary amine compound offormula:

-   -   wherein R₁ is a cyclic or polycyclic ring, wherein the ring is a        substituted or unsubstituted aryl, heteroaryl, cycloalkyl, or        heterocyclyl;    -   n=1-3;    -   wherein R₂, R₃, R₄, R₅, R₆, and R₇, are each individually        hydrogen, a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄        alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl,        heterocycloalkenyl containing from 5-6 ring atoms (wherein from        1-3 of the ring atoms is independently selected from N, NH,        N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S), C₆-C₂₄ alkaryl,        C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl, sulfhydryl,        C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀        aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀        aryloxycarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato,        carboxy, carboxylato, carbamoyl, C₁-C₂₄ alkyl-carbamoyl,        arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano,        cyanato, isocyanato, isothiocyanato, azido, formyl, thioformyl,        amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido,        C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso,        sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄        alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀        arylsulfanyl, phosphono, phosphonato, phosphinato, phospho, or        phosphino or combinations thereof,    -   wherein, R₂ and R₄ may be linked to form a cyclic or polycyclic        ring, wherein the ring is a substituted or unsubstituted aryl,        heteroaryl, cycloalkyl, or heterocyclyl; and pharmaceutically        acceptable salts thereof.

The targeting group can be conjugated directly to the thiol group of thecysteine residue of the compound or indirectly via a linker (e.g.,polyethylene glycol) prior or during the formation of nanoparticles.Depending upon the selection of the targeting group, the targeting groupcan be covalently bonded to either the thiol group of the cysteineresidues.

In one aspect, the targeting group is indirectly attached to thecompound by a linker. Examples of linkers include, but are not limitedto, a polyamine group, a polyalkylene group, a polyamino acid group or apolyethylene glycol group. The selection of the linker as well as themolecular weight of the linker can vary depending upon the desiredproperties. In one aspect, the linker is polyethylene glycol having amolecular weight from 500 to 10,000, 500 to 9,000, 500 to 8,000, 500 to7,000, or 2,000 to 5,000. In certain aspects, the targeting group isfirst reacted with the linker in a manner such that the targeting groupis covalently attached to the linker. For example, the linker canpossess one or more groups that can react with an amino group present ona targeting group. The linker also possesses additional groups thatreact with and form covalent bonds with the compounds described herein.For example, the linker can possess maleimide groups that readily reactwith the thiol groups. The selection of functional groups present on thelinker can vary depending upon the functional groups present on thecompound and the targeting group. In one aspect, the targeting group isa retinoid, such as a retinylamine (e.g., all-trans-retinylamine) orretinoid derivative, such as(1R)-3-amino-1-[3-(cyclohexylmethoxy)phenyl]propan-1-ol; hydrochloride,that is covalently attached to polyethylene glycol.

In some embodiment, the linker can include an acid labile bond, such asformed by incorporation of a hydrazone into the linker that ishydrolyzable in an endolysomal environment following uptake to cells,such as retinal or retinal pigment epithelium cells. For example, thelinker can be covalently linked to the compound by at least one of acovalent hydrolyzable ester, covalent hydrolyzable amide, covalentphotodegradable urethane, covalent hydrolyzable ester, or covalenthydrolyzable acrylate-thiol linkage. Following cellular uptake of thecompound, within the late endosomes, the increasingly acidic environmentcan cleave the acid labile linkage to promote shedding of a polymerlinker, such as PEG, and expose the core of the compound/nucleic complexnanoparticle.

In some embodiments, the compound used to form the multifunctionalpH-sensitive carriers or nanoparticles can have formula (I):

-   -   wherein R¹ is an alkylamino group or a group containing at least        one aromatic group; R² and R³ are independently an aliphatic        group or a hydrophobic group, derived, for example, from a fatty        acid;    -   R⁴ and R⁵ are independently H, a substituted or unsubstituted        alkyl group, an alkenyl group, an acyl group, or an aromatic        group, or includes a polymer, a targeting group, or a detectable        moiety and at least one of R⁴ and R⁵ includes a targeting group        that targets and/or binds to a retinal or visual protein, such        as an interphotoreceptor retinoid binding protein; a, b, c, and        d are independently an integer from 1 to 10 (e.g., a, b, c, and        d are each 2); and pharmaceutically acceptable salts thereof.

In some embodiments, R¹ can include at least one of:

-   -   where R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are        independently hydrogen, an alkyl group, a hydrophobic group, or        a nitrogen containing substituent; and e, f, g, i, j, k, and l,        are an integer from 1 to 10.

For example, R¹ can include at least one of CH₂NH₂, CH₂CH₂NH₂,CH₂CH₂CH₂NH₂, CH₂CH₂CH₂CH₂NH₂, CH₂CH₂CH₂CH₂CH₂NH₂, CH₂NHCH₂CH₂CH₂NH₂,CH₂CH₂NHCH₂CH₂CH₂NH₂, CH₂CH₂CH₂NHCH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NH₂,CH₂CH₂NHCH₂CH₂CH₂CH₂NH₂, CH₂CH₂NHCH₂CH₂CH₂NHCH₂CH₂CH₂HN₂, orCH₂CH₂NH(CH₂CH₂NH)_(d)CH₂CH₂NH₂, where d is from 0 to 10.

In some embodiments, R¹ can be CH₂CH₂NH₂ orCH₂CH₂NHCH₂CH₂CH₂NHCH₂CH₂CH₂HN₂.

In other embodiments, R² and R³ are independently an aliphatic group ora hydrophobic group derived from fatty acid, such as oleic acid orlinoleic acid, and are the same or different. The additional double bondin linoleic acid introduces an extra kink into the hydrocarbon backbone,giving the compound a broader conical shape than oleic acid andincreasing its fluidity. When incorporated into a nanoparticlestructure, the extra degree of unsaturation elevates the propensity toform the hexagonal phase during an impending membrane fusion event ofcellular uptake.

In some embodiments, at least one of R⁴ or R⁵ includes a retinoid, suchas a retinylamine (e.g., all-trans-retinylamine) or a retinoidderivative, such as(1R)-3-amino-1-[3-(cyclohexylmethoxy)phenyl]propan-1-ol; hydrochloride,that is covalently attached to a polymer linker, such as polyethyleneglycol.

The compounds having the general formula I can be synthesized usingsolid phase techniques known in the art. The Examples provides syntheticprocedures for preparing the compounds. In general, the approach in theExample involves the systematic protection/elongation/deprotection toproduce a dithiol compound. The hydrophobic group is produced byreacting oleic acid with the amino group present on the cysteineresidue. The targeting group is conjugated to a PEG spacer and thenconjugated to the compound via a Michael addition reaction. Although theExample depicts one approach for producing the compounds of formula I,other synthetic techniques can be used.

Any of the compounds described herein can exist or be converted to thesalt thereof. In one aspect, the salt is a pharmaceutically acceptablesalt. The salts can be prepared by treating the free acid with anappropriate amount of a chemically or pharmaceutically acceptable base.Representative chemically or pharmaceutically acceptable bases areammonium hydroxide, sodium hydroxide, potassium hydroxide, lithiumhydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide,zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, histidine, and the like. In oneaspect, the reaction is conducted in water, alone or in combination withan inert, water-miscible organic solvent, at a temperature of from about0° C. to about 100° C., such as at room temperature. The molar ratio ofthe compound to base used is chosen to provide the ratio desired for anysalt. For preparing, for example, the ammonium salts of the free acidstarting material, the starting material can be treated withapproximately one equivalent of base to yield a salt.

In another aspect, any of the compounds described herein can exist or beconverted to the salt with a Lewis base thereof. The compounds can betreated with an appropriate amount of Lewis base. Representative Lewisbases are ammonium hydroxide, sodium hydroxide, potassium hydroxide,lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferroushydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferrichydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, histidine, THF, ether, thiolreagent, alcohols, thiol ethers, carboxylates, phenolates, alkoxides,water, and the like. In one aspect, the reaction is conducted in water,alone or in combination with an inert, water-miscible organic solvent,at a temperature of from about 0° C. to about 100° C. such as at roomtemperature. The molar ratio of the compound to base used is chosen toprovide the ratio desired for any complex. For example, the ammoniumsalts of the free acid starting material, the starting material can betreated with approximately one equivalent of chemically orpharmaceutically acceptable Lewis base to yield a complex.

If the compounds possess carboxylic acid groups, these groups can beconverted to pharmaceutically acceptable esters or amides usingtechniques known in the art. Alternatively, if an ester is present onthe dendrimer, the ester can be converted to a pharmaceuticallyacceptable ester using transesterification techniques.

The therapeutic nucleic acid(s) that complexes with and/or is condensedby the compounds described herein to form the multifunctionalpH-sensitive carriers or nanoparticles can include any nucleic acid thatwhen introduced to or within the eye is capable of treating,ameliorating, attenuating, and/or eliminating symptoms of a disease ordisorder of the eye. The nucleic acid can be any nucleic acid encoding anatural, truncated, artificial, chimeric or recombinant product [e.g., apolypeptide of interest (including a protein or a peptide), a RNA, etc.]that is capable of treating, ameliorating, attenuating, and/oreliminating symptoms of a disease or disorder of the eye.

The nucleic acid can be a deoxyribonucleic acid (DNA) molecule (cDNA,gDNA, synthetic DNA, artificial DNA, recombinant DNA, etc.) or aribonucleic acid (RNA) molecule (mRNA, tRNA, RNAi, siRNA, catalytic RNA,antisense RNA, viral RNA, etc.). The nucleic acid may be single strandedor multistranded nucleic acid, double-stranded nucleic acid or may becomplexed. The nucleic acid may comprise hybrid sequences or syntheticor semi-synthetic sequences. It may be obtained by any technique knownto persons skilled in the art, and especially by screening libraries, bychemical synthesis, or alternatively by mixed methods including chemicalor enzymatic modification of sequences obtained by screening libraries.

In a particular embodiment, the therapeutic nucleic acid is of syntheticor biosynthetic origin or extracted from a virus or from a unicellularor pericellular eukaryotic or prokaryotic organism.

The therapeutic nucleic acid used may be naked, may be complexed withany chemical, biochemical or biological agent, may be inserted in avector, etc., when administered to the eye.

The naked DNA can refer to any nucleic acid molecule which is notcombined with a synthetic, biosynthetic, chemical, biochemical orbiological agent improving the delivery or transfer of said DNA, orfacilitating its entry into the cell.

The vector can be a nucleic acid molecule that is capable oftransporting another nucleic acid to which it has been linked. In someembodiments, the vectors are those capable of autonomous replicationand/or expression of nucleic acids to which they are linked. Vectorscapable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of “plasmids” which refer to circular double strandedDNA loops which, in their vector form, are not bound to the chromosome.In some embodiments, the plasmid can be the most commonly used form ofvector. In other embodiments, the plasmid can be a form of naked DNA asdescribed herein.

In some embodiment, the nucleic acid may also contain one or moreadditional regions, for example regulatory elements of small or largesize which are available to the skilled artisan, such as a promoterregion (constitutive, regulated, inducible, tissue-specific, etc.), forexample sequences allowing and/or promoting expression in the targetedtissue (e.g., choroid or retina) or cells (e.g., RPE or photoreceptors),a transcription termination signal, secretion sequences, an origin ofreplication and/or nuclear localization signal (nls) sequences whichfurther enhance polynucleotide transfer to the cell nucleus.

Additionally, the nucleic acid may further comprise selectable markersuseful in selecting, measuring, and monitoring nucleic acid transferresults (transfer to which tissues, duration of expression, etc.). Thetypes of expression systems and reporter genes that can be used oradapted for use are well known in the art. For example, genes coding fora luciferase activity, an alkaline phosphatase activity, or a greenfluorescent protein activity are commonly used.

The nucleic acid may contain any nucleotide sequence of any size. Thenucleic acid may thus vary in size from a simple oligonucleotide to alarger molecule such as a nucleotide sequence including exons and/orintrons and/or regulatory elements of any sizes (small or large), a geneof any size, for example of large size, or a chromosome for instance,and may be a plasmid, an episome, a viral genome, a phage, a yeastartificial chromosome, a minichromosome, an antisense molecule, etc.

In some embodiments, the nucleic acid is a double-stranded, circularDNA, such as a plasmid, encoding a product with biological activity.

The nucleic acid can be prepared and produced according to conventionalrecombinant DNA techniques, such as amplification, culture inprokaryotic or eukaryotic host cells, purification, etc. The techniquesof recombinant DNA technology are known to those of ordinary skill inthe art.

In embodiments, the therapeutic nucleic acids include nucleic acidsencoding proteins and RNAs, including siRNAs. Such therapeutic nucleicacids may act by providing an activity that is missing, or significantlyreduced, in a diseased eye. Such molecules may also act by modifying orreducing an activity that is over-expressed, or significantly elevatedabove normal levels, in a diseased eye. For example, a therapeuticnucleic acid may encode a protein possessing an activity (e.g., specificbinding activity, enzymatic activity, transcriptional regulationactivity, etc.) that is lacking in cells of the eye. Lack of suchactivity may result from failure of the cells to produce the protein,production of a mutated, inactive form of the protein, or misfolding ofa protein resulting in an inactive form. In some cases, introducing a“good” (i.e., functional) copy of the protein may alleviate symptoms ofthe disease by directly replacing the missing activity. Alternatively,therapeutic nucleic acids may act by increasing or decreasing theactivity of other proteins in cells of the eye. For example, thetherapeutic nucleic acid may encode a protein that may bind to anotherprotein and thereby either decrease or eliminate the activity of thesecond protein. Alternatively, binding of the therapeutic nucleic acidmay encode a protein that may bind to another protein in cells of theeye, which may result in stabilization of such protein and/or anincrease in the related activity. Finally, the therapeutic nucleic acidmay increase or decrease transcription of genes, or the translation oftranscripts from genes in cells of the eye. For example, a therapeuticnucleic may encode a protein that may bind to a transcriptional regionof a gene and thereby increase or decrease transcription of that gene.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level. Antisensenucleic acids can be nucleic acid fragments capable of specificallyhybridizing with a nucleic acid encoding an endogenous ocular activesubstance or the corresponding messenger RNA. These antisense nucleicacids can be synthetic oligonucleotides, optionally modified to improvetheir stability and selectivity. They can also be DNA sequences whoseexpression in the cell produces RNA complementary to all or part of themRNA encoding an endogenous ocular active substance. Antisense nucleicacids can be prepared by expression of all or part of a nucleic acidencoding an endogenous ocular active substance, in the oppositeorientation. Any length of antisense sequence is suitable for practiceof the invention so long as it is capable of down-regulating or blockingexpression of the endogenous ocular active substance. Preferably, theantisense sequence is at least 20 nucleotides in length. The preparationand use of antisense nucleic acids, DNA encoding antisense RNAs and theuse of oligo and genetic antisense is disclosed in WO92/15680, thecontent of which is incorporated herein by reference.

In some embodiments, the nucleic acid encodes or is an interfering RNA(RNAi) or antisense polynucleotide sequences useful in eliminating orreducing the production of a gene product, as described by Tso, P. et alAnnals New York Acad. Sci. 570:220-241 (1987). Typically, RNAi includeRNAi that decrease the level of an apoptotic or angiogenic factor in acell.

In some embodiments the nucleic acid is siRNA. siRNAs are doublestranded RNA molecules (dsRNAs) with approximately 20 to 25 nucleotides,which are generated by the cytoplasmic cleavage of long RNA with theRNase III enzyme Dicer. siRNAs specifically incorporate into theRNA-induced silencing complex (RISC) and then guide the RNAi machineryto destroy the target mRNA containing the complementary sequences. SinceRNAi is based on nucleotide base-pairing interactions, it can betailored to target any gene of interest, rendering siRNA an ideal toolfor treating diseases with gene silencing. Gene silencing with siRNAshas a great potential for the treatment of human diseases as a newtherapeutic modality. Numerous siRNAs have been designed and reportedfor various therapeutic purposes and some of the siRNAs havedemonstrated specific and effective silencing of genes related to humandiseases. Therapeutic applications of siRNAs include, but are notlimited to, inhibition of viral gene expression and replication inantiviral therapy, anti-angiogenic therapy of ocular diseases, treatmentof autoimmune diseases and neurological disorders, and anticancertherapy. Therapeutic gene silencing has been demonstrated in mammals,which bodes well for the clinical application of siRNA. It is believedthat siRNA can target every gene in human genome and has unlimitedpotential to treat human disease with RNAi.

For example, an RNAi can be a shRNA or siRNA that reduces the level of apolynucleotide product that induces or promotes apoptosis in a cell.Genes whose polynucleotide products induce or promote apoptosis arereferred to herein as “pro-apoptotic genes” and the products of thosegenes (mRNA; protein) are referred to as “pro-apoptotic polynucleotideproducts.” Pro-apoptotic polynucleotide products include, e.g., Bax,Bid, Bak, and Bad polynucleotide products. See, e.g., U.S. Pat. No.7,846,730. Interfering RNAs could also be against an angiogenic product,for example VEGF (e.g., Cand5; see, e.g., U.S. Patent Publication No.2011/0143400; U.S. Patent Publication No. 2008/0188437; and Reich et al.(2003) Mol. Vis. 9:210), VEGFR1 (e.g., Sirna-027; see, e.g., Kaiser etal. (2010) Am. J. Ophthalmol. 150:33; and Shen et al. (2006) Gene Ther.13:225), or VEGFR2 (Kou et al. (2005) Biochem. 44: 15064). See also,U.S. Pat. Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and5,639,736; and 7,947,659 and 7,919,473.

In some embodiments, the therapeutic nucleic acids can encodebiologically active polypeptides or proteins including enzymes, bloodderivatives, hormones, lymphokines, cytokines, chemokines,anti-inflammatory factors, growth factors, trophic factors, neurotrophicfactors, haematopoietic factors, angiogenic factors, anti-angiogenicfactors, inhibitors of metalloproteinase, regulators of apoptosis,coagulation factors, receptors thereof, in particular soluble receptors,a peptide which is an agonist or antagonist of a receptor or of anadhesion protein, antigens, antibodies, fragments or derivatives thereofand other essential constituents of the cell, proteins involved in thevisual cycle within RPE cells, and structure proteins of retinal cells(structure proteins, proteins involved in the phototransduction processand/or in the visual cycle; retinal recycling) and/or phagocytosis ofthe photoreceptor outer segment phagocytosis).

Various retina-derived neurotrophic factors have the potential to rescuedegenerating photoreceptor cells and may be delivered trough a methodaccording to the present invention. Preferred biologically active agentsmay be selected from VEGF, Angiogenin, Angiopoietin-1, DeM, acidic orbasic Fibroblast Growth Factors (aFGF and bFGF), FGF-2, Follistatin,Granulocyte Colony-Stimulating factor (G-CSF), Hepatocyte Growth Factor(HGF), Scatter Factor (SF), Leptin, Midkine, Placental Growth Factor(PGF), Platelet-Derived Endothelial Cell Growth Factor (PD-ECGF),Platelet-Derived Growth Factor-BB (PDGF-BB), Pleiotrophin (PTN), RdCVF(Rod-derived Cone Viability Factor), Progranulin, Proliferin,Transforming Growth Factor-alpha (TGF-alpha), PEDF, Transforming GrowthFactor-beta (TGF-beta), Tumor Necrosis Factor-alpha (TNF-alpha),Vascular Endothelial Growth Factor (VEGF), Vascular Permeability Factor(VPF), CNTF, BDNF, GDNF, PEDF, NT3, BFGF, angiopoietin, ephrin, EPO,NGF, IGF, GMF, aFGF, NTS, Gax, a growth hormone, [alpha]-1-antitrypsin,calcitonin, leptin, an apolipoprotein, an enzyme for the biosynthesis ofvitamins, hormones or neuromediators, chemokines, cytokines such asIL-1, IL-8, IL-10, IL-12, IL-13, a receptor thereof, an antibodyblocking any one of said receptors, TIMP such as TIMP-1, TIMP-2, TIMP-3,TIMP-4, angioarrestin, endostatin such as endostatin XVIII andendostatin XV, ATF, angiostatin, a fusion protein of endostatin andangiostatin, the C-terminal hemopexin domain of matrixmetalloproteinase-2, the kringle 5 domain of human plasminogen, a fusionprotein of endostatin and the kringle 5 domain of human plasminogen, theplacental ribonuclease inhibitor, the plasminogen activator inhibitor,the Platelet Factor-4 (PF4), a prolactin fragment, theProliferin-Related Protein (PRP), the antiangiogenic antithrombin III,the Cartilage-Derived Inhibitor (CDI), a CD59 complement fragment, C3aand C5a inhibitors, complex attack membrane inhibitors, Factor H, ICAM,VCAM, caveolin, PKC zeta, junction proteins, JAMs, CD36, MERTKvasculostatin, vasostatin (calreticulin fragment), thrombospondin,fibronectin, in particular fibronectin fragment gro-beta, an heparinase,human chorionic gonadotropin (hCG), interferon alpha/beta/gamma,interferon inducible protein (IP-10), the monokine-induced byinterferon-gamma (Mig), the interferon-alpha inducible protein 10(IP10), a fusion protein of Mig and IP10, soluble Fms-Like Tyrosinekinase 1 (FLT-1) receptor, Kinase insert Domain Receptor (KDR),regulators of apoptosis such as Bcl-2, Bad, Bak, Bax, Bik, BcI-X shortisoform and Gax, fragments or derivatives thereof and the like.

In some embodiments, the biologically active nucleic acid encodes aprecursor of a therapeutic protein such as those described above.

In some embodiments, the nucleic acid may encode for a viable protein soas to replace the defective protein which is naturally expressed in thetargeted tissue. Typically, defective genes that may be replacedinclude, but are not limited to, genes that are responsible for retinaldegenerative diseases, such as retinitis pigmentosa (RP), Lebercongenital amaurosis (LCA), recessive RP, Dominant retinitis pigmentosa,X-linked retinitis pigmentosa, Incomplete X-linked retinitis pigmentosa,dominant, Dominant Leber congenital amaurosis, Recessive ataxia,posterior column with retinitis pigmentosa, Recessive retinitispigmentosa with para-arteriolar preservation of the RPE, Retinitispigmentosa RP12, Usher syndrome, Dominant retinitis pigmentosa withsensorineural deafness, Recessive retinitis Punctata albescens,Recessive Alstrom syndrome, Recessive Bardet-Biedl syndrome, Dominantspinocerebellar ataxia w/macular dystrophy or retinal degeneration,Recessive abetalipoproteinemia, Recessive retinitis pigmentosa withmacular degeneration, Recessive Refsum disease, adult form, RecessiveRefsum disease, infantile form, Recessive enhanced S-cone syndrome,Retinitis pigmentosa with mental retardation, Retinitis pigmentosa withmyopathy, Recessive Newfoundland rod-cone dystrophy, Retinitispigmentosa sinpigmento, Sector retinitis pigmentosa, Regional retinitispigmentosa, Senior-Loken syndrome, Joubert syndrome, Stargardt disease,juvenile, Stargardt disease, late onset, Dominant macular dystrophy,Stargardt type, Dominant Stargardt-like macular dystrophy, Recessivemacular dystrophy, Recessive fundus flavimaculatus, Recessive cone-roddystrophy, X-linked progressive cone-rod dystrophy, Dominant cone-roddystrophy, Cone-rod dystrophy; de Grouchy syndrome, Dominant conedystrophy, X-linked cone dystrophy, Recessive cone dystrophy, Recessivecone dystrophy with supernormal rod electroretinogram, X-linked atrophicmacular dystrophy, X-linked retinoschisis, Dominant macular dystrophy,Dominant radial, macular drusen, Dominant macular dystrophy, bull's-eye,Dominant macular dystrophy, butterfly-shaped, Dominant adult vitelliformmacular dystrophy, Dominant macular dystrophy, North Carolina type,Dominant retinal-cone dystrophy 1, Dominant macular dystrophy, cystoid,Dominant macular dystrophy, atypical vitelliform, Foveomacular atrophy,Dominant macular dystrophy, Best type, Dominant macular dystrophy, NorthCarolina-like with progressive, Recessive macular dystrophy, juvenilewith hypotrichosis, Recessive foveal hypoplasia and anterior segmentdysgenesis, Recessive delayed cone adaptation, Macular dystrophy in bluecone monochromacy, Macular pattern dystrophy with type II diabetes anddeafness, Flecked Retina of Kandori, Pattern Dystrophy, DominantStickler syndrome, Dominant Marshall syndrome, Dominant vitreoretinaldegeneration, Dominant familial exudative vitreoretinopathy, Dominantvitreoretinochoroidopathy; Dominant neovascular inflammatoryvitreoretinopathy, Goldmann-Favre syndrome, Recessive achromatopsia,Dominant tritanopia, Recessive rod monochromacy, Congenital red-greendeficiency, Deuteranopia, Protanopia, Deuteranomaly, Protanomaly,Recessive Oguchi disease, Dominant macular dystrophy, late onset,Recessive gyrate atrophy, Dominant atrophia greata, Dominant centralareolar choroidal dystrophy, X-linked choroideremia, Choroidal atrophy,Central areolar, Central, Peripapillary, Dominant progressive bifocalchorioretinal atrophy, Progresive bifocal Choroioretinal atrophy,Dominant Doyne honeycomb retinal degeneration (Malattia Leventinese),Amelogenesis imperfecta, Recessive Bietti crystalline corneoretinaldystrophy, Dominant hereditary vascular retinopathy with Raynaudphenomenon and migraine, Dominant Wagner disease and erosivevitreoretinopathy, Recessive microphthalmos and retinal diseasesyndrome; Recessive nanophthalmos, Recessive retardation, spasticity andretinal degeneration, Recessive Bothnia dystrophy, Recessivepseudoxanthoma elasticum, Dominant pseudoxanthoma elasticum; RecessiveBatten disease (ceroid-lipofuscinosis), juvenile, Dominant Alagillesyndrome, McKusick-Kaufman syndrome, hypoprebetalipoproteinemia,acanthocytosis, palladial degeneration; Recessive Hallervorden-Spatzsyndrome; Dominant Sorsby's fundus dystrophy, Oregon eye disease,Kearns-Sayre syndrome, Retinitis pigmentosa with developmental andneurological abnormalities, Basseb Korenzweig Syndrome, Hurler disease,Sanfilippo disease, Scieie disease, Melanoma associated retinopathy,Sheen retinal dystrophy, Duchenne macular dystrophy, Becker maculardystrophy, and Birdshot Retinochoroidopathy. Examples of genes includebut are not limited to genes encoding for ATP-binding cassettetransporter (e.g., ABCA4), RPE65, RHO, RdCVF, CP290.

In other embodiments, the nucleic acid encodes a site-specificendonuclease that provides for site-specific knock-down of genefunction, e.g., where the endonuclease knocks out an allele associatedwith a retinal disease. For example, where a dominant allele encodes adefective copy of a gene that, when wild-type, is a retinal structuralprotein and/or provides for normal retinal function, a site-specificendonuclease (such as TALEnucleases, meganucleases or Zinc fingernucleases) can be targeted to the defective allele and knock out thedefective allele. In addition to knocking out a defective allele, asite-specific nuclease can also be used to stimulate homologousrecombination with a donor DNA that encodes a functional copy of theprotein encoded by the defective allele. Thus, e.g., the method of theinvention can be used to deliver both a site-specific endonuclease thatknocks out a defective allele, and can be used to deliver a functionalcopy of the defective allele, resulting in repair of the defectiveallele, thereby providing for production of a functional retinal protein(e.g., functional retinoschisin, functional RPE65, functionalperipherin, etc.). See, e.g., Li et al. (2011) Nature 475:217. In someembodiments, the vector comprises a polynucleotide that encodes asite-specific endonuclease; and a polynucleotide that encodes afunctional copy of a defective allele, where the functional copy encodesa functional retinal protein. Functional retinal proteins include, e.g.,retinoschisin, RPE65, retinitis pigmentosa GTPase regulator(RGPR)-interacting protein-1, peripherin, peripherin-2, and the like.Site-specific endonucleases that are suitable for use include, e.g.,zinc finger nucleases (ZFNs); and transcription activator-like effectornucleases (TALENs), where such site-specific endonucleases arenon-naturally occurring and are modified to target a specific gene. Suchsite-specific nucleases can be engineered to cut specific locationswithin a genome, and non-homologous end joining can then repair thebreak while inserting or deleting several nucleotides. Suchsite-specific endonucleases (also referred to as “INDELs”) then throwthe protein out of frame and effectively knock out the gene. See, e.g.,U.S. Patent Publication No. 2011/0301073.

The nucleic acid can be complexed to the carrier compounds describedherein by admixing the nucleic acid and the compound or correspondingdisulfide oligomer or polymer. The pH of the reaction can be modified toconvert the amino groups present on the compounds described herein tocationic groups. For example, the pH can be adjusted to protonate theamino group. With the presence of cationic groups on the compound, thenucleic acid can electrostatically bond (i.e., complex) to the compound.In one aspect, the pH is from 1 to 7.4. In another aspect, the N/P ratiois from 0.5 to 100, where N is the number of nitrogen atoms present onthe compound that can be form a positive charge and P is the number ofphosphate groups present on the nucleic acid. Thus, by modifying thecompound with the appropriate number of amino groups in the head group,it is possible to tailor the bonding (e.g., type and strength of bond)between the nucleic acid and the compound. The N/P ratio can be adjusteddepending on the cell type to which the nucleic acid is to be delivered.In some embodiments where the cell is cancer, the N/P ratio can be atleast about 6, at least about 10, or at least about 15. In otherembodiments, the N/P ration can be from about 6 to about 20.

In one aspect, the nucleic acid/carrier complex is a nanoparticle. Inone aspect, the nanoparticle has a diameter of about 1000 nanometers orless.

In other aspects, the compounds described herein can be designed so thatthe resulting nucleic acid nanoparticle escapes endosomal and/orlysosomal compartments at the endosomal-lysosomal pH. For example, thecompound forming nanoparticles with nucleic acids can be designed suchthat its structure and amphiphilicity changes at endosomal-lysosomal pH(5.0-6.0) and disrupts endosomal-lysosomal membranes, which allows entryof the nanoparticle into the cytoplasm. In one aspect, the ability ofspecific endosomal-lysosomal membrane disruption of the compoundsdescribed herein can be tuned by modifying their pH sensitiveamphiphlicity by altering the number and structure of protonatableamines and lipophilic groups. For example, decreasing the number ofprotonatable amino groups can reduce the amphiphilicity of ananoparticle produced by the compound at neutral pH. In one aspect, thecompounds herein have 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to8, 1 to 6, 1 to 4, or 2 protonatable amino or substituted amino groups.The pH-sensitive amphiphilicity of the compounds and nanoparticlesproduced by the compounds can be used to fine-tune the overall pKa ofthe nanoparticle. Low amphiphilicity of the nanoparticles atphysiological pH can minimize non-specific cell membrane disruption andnonspecific tissue uptake of the nucleic acid/MFC system. In certainaspects, it is desirable that the carriers have low amphiphilicity atthe physiological pH and high amphiphilicity at the endosomal-lysosomalpH, which will only cause selective endosomal-lysosomal membranedisruption with the nanoparticles.

The surface of the nanoparticle complexes can be modified by, forexample, covalently incorporating polyethylene glycol by reactingunpolymerized free thiol of the nanoparticle to reduce non-specifictissue uptake in vivo. For example, PEG-maleimide reacts rapidly withfree thiol groups. The molecular weight of the PEG can vary dependingupon the desired amount of hydrophilicity to be imparted on the carrier.PEG-modification of the carrier can also protect nanoparticles composedof the nucleic acid from enzymatic degradation upon uptake by the cell(e.g., endonucleases).

In some embodiments, the amount or mole percent of the targeting groupsprovided on or attached to the surface of the nanoparticle can be about1 mol % to about 10 mol % of the compounds that form the nanoparticle,for example, about 1 mol % to about 5 mol % (e.g., about 2.5 mol %) ofthe compounds that form the nanoparticle.

Advantageously, multifunctional pH-sensitive carriers formed using thecompounds have improved stability when administered systemically to asubject, protect condensed nucleic acids from degradation, and promoteendosomal escape and cytosolic release upon cellular uptake.

The compounds described herein can be used in a method to introduce anucleic acid into a cell of the eye or a cell delivered to the eye. Themethod generally involves contacting the cell with a complex, whereinthe nucleic acid is taken up into the cell. In one aspect, the compoundsdescribed herein can facilitate the delivery of DNA or RNA as therapyfor genetic disease by supplying deficient or absent gene products totreat any genetic disease or by silencing gene expression. Techniquesknown in the art can used to measure the efficiency of the compoundsdescribed herein to deliver nucleic acids to a cell.

In some embodiments, the cell can be a cell within the eye. Examples ofcells within the eye can include cells located in the ganglion celllayer (GCL), the inner plexiform layer inner (IPL), the inner nuclearlayer (INL), the outer plexiform layer (OPL), outer nuclear layer (ONL),outer segments (OS) of rods and cones, the retinal pigmented epithelium(RPE), the inner segments (IS) of rods and cones, the epithelium of theconjunctiva, the iris, the ciliary body, the corneum, and epithelium ofocular sebaceous glands.

The complexes (i.e., nanoparticles) described above can be administeredto a subject using techniques known in the art. For example,pharmaceutical compositions can be prepared with the complexes. It willbe appreciated that the actual preferred amounts of the complex in aspecified case will vary according to the specific compound beingutilized, the particular compositions formulated, the mode ofapplication, and the particular sites and subject being treated. Dosagesfor a given host can be determined using conventional considerations,e.g., by customary comparison of the differential activities of thesubject compounds and of a known agent, e.g., by means of an appropriateconventional pharmacological protocol. Physicians and formulators,skilled in the art of determining doses of pharmaceutical compounds,will have no problems determining dose according to standardrecommendations (Physicians Desk Reference, Barnhart Publishing (1999).

Pharmaceutical compositions described herein can be formulated in anyexcipient the biological system or entity can tolerate. Examples of suchexcipients include, but are not limited to, water, saline, Ringer'ssolution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, vegetable oils such as olive oil and sesame oil,triglycerides, propylene glycol, polyethylene glycol, and injectableorganic esters such as ethyl oleate can also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosol, cresols, formalin and benzyl alcohol.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration to humans,including solutions such as sterile water, saline, and bufferedsolutions at physiological pH.

Molecules intended for pharmaceutical delivery can be formulated in apharmaceutical composition. Pharmaceutical compositions can includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more activeingredients such as antimicrobial agents, antiinflammatory agents,anesthetics, and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration can be topically, includingophthalmically. In the case of contacting cells with the nanoparticlecomplexes of nucleic acid described herein, it is possible to contactthe cells in vivo or ex vivo.

In some embodiments, the complexes (i.e., nanoparticles) areadministered to an eye of the subject such that they are able totraverse the outer layers of the eye (i.e., cornea, iris, sclera, pupil,lens, or conjunctiva) and enter into the intraocular fluid (alsoreferred to as the aqueous humor). In other embodiments, the complexes(i.e., nanoparticles) are able to traverse the outer layers of the eyeand enter into the intraocular fluid. Thus, in certain embodimentscomplexes (i.e., nanoparticles) are administered topically to the eye.In some embodiments, the complexes are injected into the eye. This mayinclude intramuscular, intradermal, subcutaneous, subconjunctival andsub-Tenon's, intravitreal, subretinal, intravenous and intracameralinjections. Such injections can deliver the complexes to the intraocularfluid or to a location within the retina. In one embodiment, theinjection delivers the complexes to the intraocular fluid. In oneembodiment, the injection delivers the complexes into the retina. In oneembodiment, the complexes are administered by intravitreal injection. Inanother embodiment, the complexes are administered by subretinalinjection. In another embodiment, the complexes are administered bysub-Tenon's injection. Methods of performing intraocular injections areknown to those skilled in the art.

Preparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles, if needed forcollateral use of the disclosed compositions and methods, include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles, if needed forcollateral use of the disclosed compositions and methods, include fluidand nutrient replenishers, electrolyte replenishers (such as those basedon Ringer's dextrose), and the like. Preservatives and other additivescan also be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until one ofordinary skill in the art determines the delivery should cease. Personsof ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. It is understood that any givenparticular aspect of the disclosed compositions and methods can beeasily compared to the specific examples and embodiments disclosedherein, including the reagents discussed in the Examples. By performingsuch a comparison, the relative efficacy of each particular embodimentcan be easily determined. Particularly preferred compositions andmethods are disclosed in the Examples herein, and it is understood thatthese compositions and methods, while not necessarily limiting, can beperformed with any of the compositions and methods disclosed herein.

The following Examples are for the purpose of illustration only and isnot intended to limit the scope of the claims, which are appendedhereto.

Example 1

In this Example, we designed and prepared all-trans-retinylaminemodified ECO plasmid DNA (pDNA) nanoparticles with PEG spacer to targetinterphotoreceptor retinoid-binding protein (IRBP) for enhanced genedelivery in the retina. All-trans-retinoids have a high binding affinityfor retinoid binding proteins, which play important roles in visualtransduction. IRBP is a major protein in the interphotoreceptor matrix(IPM), and selectively transports 11-cis-retinal to the photoreceptorouter segment and all-trans-retinol to the RPE. This selective transportmechanism can increase the transfection efficiency directly to the RPEwith the ECO/pDNA nanoparticles conjugated with all-trans-retinylamine.The in vitro transfection efficiency of the ECO/pDNA nanoparticles wasevaluated in ARPE-19, a human RPE cell line. The in vivo transfectionefficiency of the targeted ECO/pDNA nanoparticles to the RPE was thenevaluated in the wild type BABL/c mice using GFP plasmids. The efficacyof gene therapy of the targeted nanoparticles was determined by ERG inthe rpe65^(−/−) mouse model of LCA2.

Materials and Methods Cell Culture

ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium andsupplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and100 unites/mL penicillin (all reagents were from Invitrogen, Waltham,Mass.). Cells were maintained in a humidified incubator at 37° C. and 5%CO₂.

Animal

BALB/c wild type mice were obtained from Jackson Laboratory (Bar Harbor,Me.). rpe65^(−/−) deficient C57BL6 mice were obtained from MichaelRedmond (National Eye Institute, National Institutes of Health,Bethesda, Md.) and genotyped as described previously. All mice werehoused and cared for in the animal facility at the School of Medicine,Case Western Reserve University, and animal procedures were approved byCWRU Institutional Animal Care and Use Committee.

Synthesis of Ret-PEG3.4k

Ligand Ret-PEG-3.4k was synthesized through a one-pot reaction.All-trans-retinylamine (15 mg) and MAL-PEG3.4k-SCM (NANOCS, New York,N.Y.) (55 mg) were added to 15 mL dimethylformamide (DMF). The solutionwas stirred at room temperature overnight. After reaction, productRet-PEG-3.4k was precipitated in 50 mL diethyl ether and washed for 3times. The product was dried under vacuum to yield yellow powderRet-PEG-3.4k. The yield is 89%.

Preparation of ECO/pDNA and Ret-PEG3.4k-ECO/pDNA Nanoparticles

Multifunctional pH-sensitive lipid ECO were synthesized as previouslyreported. The ECO/pDNA nanoparticles were prepared by a stepwiseself-assembly of ECO with plasmid DNA at an amine/phosphate (N/P) ratioof 6. The ECO stock solution (2.5 mM in ethanol) and plasmid DNA stocksolution (0.5 mg/mL) at predetermined amounts based on the N/P ratiowere diluted into equal volumes with nuclease-free water, mixed andshaken for 30 min at room temperature. The Ret-PEG3.4k solution (0.4 mMin 50% DMSO and water) was then added to the mixture at 2.5 mol % andshaken for another 30 min for the reaction to occur between maleimidefunctional group and free thiols on ECO. A different ECO stock solution(25 mM) was used for in vivo formulations. Lipofectamine 2000(Invitrogen, Waltham, Mass.)/DNA nanoparticles were prepared accordingto the manufacturer's recommendation.

Transmission Electron Microscope

The morphology of ECO/pDNA (N/P=6/1) and Ret-PEG3.4k-ECO/pDNA (N/P=6/1)nanoparticles were checked with a transmission electron microscope (JEOLJEM2200FS). Samples for TEM were prepared by depositing 20 μL of theparticle solution onto a 300-mesh copper grid covered by a thinamorphous carbon film (20 nm). Immediately after deposition, the excessliquid was removed by touching the grid with filter paper. Samples werestained twice by the addition of 3 μL of 2% uranyl acetate aqueoussolution. The excess of staining solution was removed again. Sampleswere dried and images were taken right after.

In Vitro Transfection

ARPE-19 cells were seeded onto 12-well plates at a density of 4×10⁴cells per well and allowed to grow for 24 h at 37° C. Transfections wereconducted in 10% serum media with the nanoparticles of GFP plasmid DNA(Altogen Biosystems, Las Vegas, Nev.) (catalog number 4060) at a DNAconcentration of 1 μg/mL. ECO/pGFP nanoparticles were incubated withARPE-19 cells for 8 hours at 37° C. The media then was replaced withfresh serum-containing media (10% serum) and cells were then culturedfor an additional 48 h. GFP expression was monitored with an OlympusFV1000 confocal microscope (Olympus, Center Valley, Pa.). After theculture media was removed, each well was washed twice with PBS (10 mMsodium phosphate, pH 7.2, and 100 mM NaCl). Cells were harvested aftertreatment with 0.25% trypsin containing 0.26 mM EDTA (Invitrogen),followed by centrifugation at 1000 rpm for 5 min, fixation in 750 μL PBScontaining 4% paraformaldehyde, and finally passed through a 35 μm cellstrainer (BD Biosciences, San Jose, Calif.). A BD FACSCalibur flowcytometer (BD Biosciences) was used to determine GFP expression based onthe fluorescence intensity in a total of 10,000 cells for each sample.

Intracellular Trafficking

ARPE-19 cells (4×10⁴/well) were seeded onto glass-bottom micro-welldishes and allowed to grow for 24 h at 37° C. The cells were stainedwith 4 μg/mL Hoechst 33342 (Invitrogen) and 100 mM LysoTracker Green(Life Technologies, Carlsbad, Calif.). Cells were then treated withECO/Cy3-pDNA (Mirus Bio LLC, Madison, Wis.) (catalog number MIR7904)(N/P ratio 6/1) nanoparticles in 10% serum media. Cells were culturedwith nanoparticles for 1 h, 4 h and 24 h (media was replaced by freshmedia after 4 h), when the medium was removed and they were washed withPBS for three times before fixation with PBS containing 4%paraformaldehyde. Fluorescence images were taken with an Olympus FV1000confocal microscope.

In Vivo Subretinal Transfection with ECO/pDNA and Ret-PEG3.4k-ECO/pDNANanoparticles

All surgical manipulations were carried out under a surgical microscope(Leica M651 MSD). Mice were anesthetized by intraperitoneal injection of20 μL/g of body weight of 6 mg/mL ketamine and 0.44 mg/mL xylazine in 10mM sodium phosphate and 100 mM NaCl buffer solution (pH=7.2). Pupilswere dilated with 1.0% tropicamide ophthalmic solution (Bausch & Lomb,Rochester, N.Y.). A 33-gauge beveled needle (World PrecisionInstruments, Sarasota, Fla.) was used as a lance to make a fullthickness cut through sclera at 1.0 mm posterior to the limbus. Thisneedle was replaced with a 36-gauge beveled needle attached to aninjection system (UMP-II microsyringe pump and a Micro4 controller witha footswitch, World Precision Instruments). This needle was aimed towardthe inferior nasal area of the retina, and either an ECO/pDNA orRet-PEG3.4k-ECO/pDNA nanoparticles solution (2 μL) was injected at apRPE65 (Origene, Rockville, Md.) (catalog number SC119977) or pGFP doseof 200 ng into the subretinal space. Successful administration wasconfirmed by observing bleb formation. The tip of the needle remained inthe bleb for 10 s after bleb formation, when the needle was gentlywithdrawn. A solution (2 μL) of Ret-PEG3.4k-ECO carrier alone with thesame concentration as Ret-PEG3.4k-ECO/pDNA nanoparticles was alsoinjected into the subretinal space of the contra eye served as a control(vehicle injected). To check GFP expression 3 days after injection, eyeswere collected, washed with penicillin-streptomycin solution (Sigma),and rinsed with Hanks' balanced salt solution (Hyclone, Waltham, Mass.).Eye cups were prepared as previously described. The retina and RPElayers were placed in glass bottom confocal plate and fixed with 1 mL ofPBS with 4% paraformaldehyde. An Olympus FV1000 confocal microscope wasused to assess GFP expression as noted above.

Quantitative RT-PCR (qRT-PCR)

Total RNA was isolated from the eyes of rpe65^(−/−) mice 15 days aftersubretinal injection with Ret-PEG3.4k-ECO/pRPE65 nanoparticles. cDNA wassynthesized with the QuantiTect Reverse Transcription Kit (Qiagen)following the manufacturer's instructions. Quantitative RT-PCRamplification was performed using SYBR Green I Master mix (RocheDiagnostics, Risch-Rotkreuz, Switzerland). Fold changes were calculatedafter normalizing the data to Glyceraldehyde 3-phosphate dehydrogenase.Rpe65^(−/−) mice with no treatment were used as control group.

Electroreinograms

Electroretinograms were acquired as previously described. Animals wereanesthetized by intraperitoneal injection of a cocktail (15 μL/g bodyweight) comprised of ketamine (6 mg/mL) and xylazine (0.44 mg/mL) in PBSbuffer (10 mM sodium phosphate, pH 7.2, and 100 mM NaCl). Pupils weredilated with 1% tropicamide for imaging. Experiments were performed in adark room. Three electrodes were placed on the animal: a contact lenselectrode on the eye, a reference electrode underneath the skin betweenthe ears, and a ground electrode underneath the skin of the tail.Electroretinograms were recorded with the universal electrophysiologicsystem UTAS E-3000 (LKC Technologies, Inc., Gaithersburg, Md., USA).Light intensity calibrated by the manufacturer was computer-controlled.

Histology

The eye cups for histology were fixed in 2% glutaraldehyde, 4%paraformaldehyde and processed for visualization by OCT (optimum cuttingtemperature formulation). Sections were cut at 1 μm. Slides samples werepermeabilized and fixed sequentially with 4% PFA and 0.25% Triton X-100followed by blocking with 0.5% BSA blocking solution for 1 h at roomtemperature. PNA-lectin were applied at a concentration of 12.5 μg/mLfor 1 h at room temperature and washed 3 times with a 0.1% TBST(tris-buffered saline with tween 20) for 5 min each wash. Slides werecounter-stained with DAPI and mounted with coverslip using the ProlongGold regent (Invitrogen) before imaging. Stained tissue was imaged withan Olympus FV1000 confocal microscope.

Statistical Analysis

Experiments were performed in triplicate and presented as the means andstandard deviations. Statistical analysis was conducted with two-tailedStudent's t-tests using a 95% confidence interval. Statisticalsignificance was accepted when p<0.05.

Results In Vitro Transfection of ECO/pDNA Nanoparticles

To determine the transfection efficiency of ECO in vitro, human RPEcells (ARPE-19) were transfected with ECO/pGFP (N/P=6/1) nanoparticles,and GFP expression was determined 48 h after transfection by confocalmicroscopy (FIGS. 1A, B). ECO/pGFP nanoparticles produced significantGFP expression with 69.7% cells expressing GFP, while the controllipofectamine transfected only 14.4% cells as determined by flowcytometry (FIG. 1B). High gene expression efficiency of ECO/pDNAnanoparticles was correlated to their efficient intracellular uptake.FIG. 1C shows the intracellular uptake of ECO/pDNA nanoparticles asimaged by 3D confocal microscopy at 1, 4 and 24 h post transfection withCy3-pDNA as the tracker. After 1 h incubation, ECO/Cy3-pDNAnanoparticles (red) were aligned at the surface of the cell membrane,due to their positively charged electrostatic interactions with thenegatively charged cell membrane. After 4 h, the nanoparticles enteredthe cell and co-localized with late endosomes, indicated by the yellowcolor. After 24 h, almost all the nanoparticles escaped endosomalentrapment, shown by the red fluorescence in the cytoplasm and thediminished overlap with the endosomes. The efficient cytosolic pDNAdelivery of ECO/pDNA nanoparticles resulted in high gene expressionefficiency in the RPE cells in vitro.

Preparation of Retinylamine Targeted ECO/pDNA Nanoparticles

To target IRBP, an all-trans-retinoid structure was introduced into thesurface of ECO/pDNA nanoparticles via a PEG (3.4) spacer.All-trans-retinylamine (Ret-NH₂) was first reacted with the NHSactivated ester of NHS-PEG-Mal to yield a Ret-PEG-Mal, which wasconfirmed by MALDI-TOF mass spectroscopy (FIGS. 2A, B). To form targetedECO/pDNA nanoparticles, Ret-PEG-Mal first reacted with the 2.5 mol-% ECOvia Michael addition between the thiol and maleimide. The targetednanoparticles were then formed by self-assembly with pDNA. FIGS. 2C, Dshow the transmission electron microscopic images of the ECO/pDNAnanoparticles and targeted ECO/pDNA nanoparticles. The average size ofECO/pDNA nanoparticles was approximately 100 nm. The average size ofRet-PEG-ECO/pDNA nanoparticles to around 120 nm, a slight increase aftersurface modification with Ret-PEG.

In Vivo Transfection with Targeted Ret-PEG-ECO/pGFP Nanoparticles inWild Type BALB/c Mice

The Ret-PEG-ECO/pGFP nanoparticles were subretinally injected in BALB/cmice to determine in vivo gene delivery and expression efficiency withGFP as a reporter gene. Significant GFP expression was observed in theRPE flatmounts with both unmodified ECO/pGFP nanoparticles and targetedRet-PEG-ECO/pGFP nanoparticles 3-days post injection. However,Ret-PEG-ECO/pGFP nanoparticles produced greater GFP expression thanECO/pGFP nanoparticles (FIG. 3A). ZO-1 staining of tight junctionproteins in RPE flatmounts further confirmed that the enhanced GFPexpression emanated from RPE cells (FIG. 3B).

Gene Replacement Therapy Using Ret-PEG-ECO/pRPE65 Nanoparticles inRpe65^(−/−) Mice

Gene therapy with all-trans-retinylamine modified ECO nanoparticles wasconducted in rpe65^(−/−) mice, in which RPE65 was completely knockeddown. The rpe65^(−/−) mice exhibited the phenotypic features similar tohuman LCA2 patients. Ret-PEG-ECO/pRPE65 nanoparticles were injected intothe subretinal space of 1-month-old rpe65^(−/−) mice. At 15-days postinjection, the treatment with Ret-PEG-ECO/pRPE65 nanoparticle producedhigher mRNA levels in the treated group than in the untreated controlgroup (FIG. 4A). This finding demonstrates the successful introductionof the therapeutic gene. Electroretinography (ERG) was performed at theintensity of 1.6 log cd×s/m² to determine the efficacy of thenanoparticle treatment based on the electrical responses to light fromthe retina. FIG. 4B shows a significant scotopic and photopic ERGresponse waveforms in the nanoparticle treatment group at 7 dayspost-treatment, whereas there was almost no response in control groupinjected with Ret-PEG-ECO. The amplitudes of the major waves from allERG tests were calculated at 3, 7, 30, and 120 days post-treatment(FIGS. 4C-F). Significant increase in amplitudes of scotopic a-waves andb-waves were observed for nanoparticle treatment groups, but not forcontrol groups (vehicle injected). The introduction of exogenous RPE65gene increased about 50% of the scotopic ERG amplitude throughout alltime points up to 120 days (FIGS. 4C, E), which demonstrated an improvedfunction of rod photoreceptors. The cone function also improved,represented by a five-fold increase in photopic b-wave amplitude after 3days and a three-fold increase after 7 days. Although the amplitudedecreased with later time points, the photopic b-wave amplitude of thetreatment group was double that of the control, even at 120 days.Photopic a-waves were higher in the treatment groups than in thecontrols, but the difference was not statistically significant.

Cone Preservation after Gene Replacement Therapy UsingRet-PEG-ECO/pRPE65 Nanoparticles in rpe65^(−/−) Mice

To determine whether Ret-PEG-ECO/pRPE65 nanoparticles could recue conecells in rpe65⁻⁷ mice, cryo-sections of the whole retina were preparedat 120 post-injection and cone cells were stained with peanut agglutinin(green). Compared with the control group (FIG. 5A), the treatment group(FIG. 5B) showed substantial green fluorescence staining, whichrepresented a greater number of healthy cone photoreceptors. This resultalso explained the increase of photopic wave amplitudes in ERG.Interestingly, fewer cone cells were observed away from injection site(FIG. 5C), suggesting a local rescue in this gene therapy approach.

Therapeutic Effect of Gene Replacement Therapy Using Ret-PEG-ECO/pRPE65Nanoparticles in 3-month-old rpe65^(−/−) Mice

In order to determine the optimal timing for gene replacement therapy ofLCA2 with the targeted nanoparticles, we initiated RPE65 gene therapywith Ret-PEG-ECO/pRPE65 nanoparticles in 3-month-old rpe65^(−/−) mice,and ERG tests were performed to evaluate therapeutic efficacy. Accordingto the ERG responses measured at 7 and 30 days post-treatment, nodifferences were observed for scotopic and photopic waveforms betweenthe treatment and control groups (FIG. 6), demonstrating no observableimprovement of eye function. The result suggest that gene replacementtherapy with the targeted nanoparticles in these older mice was not aseffective in restoring vision as in the younger mice, likely due to theprogression of irreversible retinal degeneration in the former agegroup.

Safety Assessment of Ret-PEG-ECO/pRPE65 Nanoparticles in BALB/c Mice

To evaluate the safety of Ret-PEG-ECO/pRPE65 nanoparticles in genetherapy, the nanoparticles were injected into the subretinal space ofhealthy 1-month-old BALB/c mice and ERG tests were conducted at 7 and 30post-injection. ERG responses for both the nanoparticle injected groupand the un-injected group were comparable at each light intensity (FIG.7A). A slight decrease in response amplitudes was observed for somemajor waveforms at 7 days because of the inflammation introduced bysubretinal injection. Eye function after nanoparticle injection becamenormal at 30 days and no deleterious effects were introduced in the ERGmajor wave amplitudes (FIG. 7B-E). The result indicates thatRet-PEG-ECO/pRPE65 nanoparticles are safe for subretinal injection ingene replace therapy.

Example 2

In this Example, ACU4429 (Emixustat), a stable analogue ofall-trans-retinylamine, which binds to the retinal pigment epithelium 65protein (RPE65) to inhibit the retinoid cycle, was introduced as atargeting ligand to achieve specific and efficient gene delivery to theRPE by conjugating the ECO-based gene delivery platform with a PEGspacer and a pH-sensitive hydrazone linker (ACU-PEG-HZ). ACU4429targeted dual pH-sensitive ECO/pDNA nanoparticles (ACU-PEG-HZ-ECO/pDNA)were prepared by self-assembly of the ACU-PEG-HZ ligand with ECO andplasmid DNA. A plasmid DNA expressing ABCA4 protein was used as a largetherapeutic gene to test the specificity and efficacy of the targetednanoparticles. The ABCA4 protein is the flippase located inphotoreceptor outer segments that helps the elimination ofretinaldehyde, a toxic photoproduct from the visual cycle. Mutations inthe ABCA4 gene can cause Stargart disease (STGD) and cone-roddystrophies. A recent study has shown that ABCA4 is detected in the RPEof mice, and genetically modified mice with ABCA4 expression only in theRPE, and not in photoreceptor cells, demonstrated a partial rescueeffect of photoreceptor degeneration in Abca4^(−/−) mice, suggestingthat gene therapy to enhance ABCA4 expression in the RPE could preventphotoreceptor degeneration in Abca4^(−/−) mice and possibly in patientswith ABCA4 mutations. RPE-specific delivery and gene expression ofACU-PEG-HZ-ECO/pDNA nanoparticles was assessed both in vitro and in vivowith ABCA4 plasmids of different promoters.

Materials and Methods Reagents

All reagents ordered from vendors were directly used without extrapurification unless they were otherwise detailed in this section.Organic solvents such as Methylene chloride (DCM), acetonitrile (ACN)chloroform, and methanol were ordered from Thermo Fisher Scientific(Hampton, N.H.). NHS-PEG-SH (MW 3400) was purchased from Nanocs lnc (NewYork, N.Y.). The synthesis of Lipid ECO followed the procedures reportedpreviously (38). For cell culture, penicillin fetal bovine serum, andstreptomycin were purchased from Invitrogen (Carlsbad, Calif.). TheABCA4 plasmid (pCMV-ABCA4) was kindly gifted by Dr. Robert S. Molday(University of British Columbia), which included human ABCA4 cDNAsequence of full-length (NCBI Accession #NM_000350.2) on a pCEP4backbone. pRHO-ABCA4 was prepared as previously described (20). Cy3-pDNAwas purchased from Mirus Bio (catalog number MIR7904). ACU-4429 was agift from Dr. Krzysztof Palczewski (University of California, Irvine).

Cell Culture

ARPE-19 (ATCC, Manassas, Va.) cells were passaged and maintained in aDulbecco's modified Eagle's medium containing fetal bovine serum (10%),streptomycin (100 μg/mL), and penicillin (100 units/mL). Cells were keptin a humidified incubator at 37° C. and 5% CO₂.

Animal

Pigmented Abca4^(−/−) knockout mice were obtained as describedpreviously and maintained with mixed backgrounds of 129Sv/Ev or C57BL/6.Animals were housed and bred in the Animal Resource Center at CWRU. Allprocedures followed approved protocols by the CWRU Institutional AnimalCare and Use Committee (IACUC #2014-0053), which were also in compliancewith recommendations from the Association for Research for Vision andOphthalmology and the American Veterinary Medical Association Panel onEuthanasia.

Synthesis of ACU-PEG-HZ-MAL

ACU-PEG-HZ-MAL was prepared following multi-step reactions. ACU4429 (25mg) was dissolved in DMF (5 mL) and 1 mL of NHS-PEG3400-SH (70.4 mg) inDMF was added drop-wise to the mixture, followed by the addition ofDIPEA (100 μL). After stirring at room temperature for 4 hours, themixture was added dropwise to diethyl ether (3× volume excess) to removeunreacted ACU4429. ACU-PEG3400-SH was obtained as precipitate in ether.To fully reduce any disulfide bond present in the synthesizedACU-PEG-SH, dithiothreitol (100 mg) was added to a solution of thesynthesized ligand and stirred overnight. A desalting spin column (1.8KMWCO) (Thermo Fisher Scientific, Waltham, Mass.) was used to remove freedithiothreitol. The product was lyophilized, and further characterizedby MALDI-TOF mass spectrometry (M_(w) increase of 160 was observed).ACU-PEG-Hydrazide was synthesized by the reaction of ACU-PEG-SH (25.2mg) and N-ε-maleimidocaproic acid hydrazide (4.25 mg) in the presence oftriethylamine (4.39 μL) mixed in a 5 mL chloroform solution for 4 hoursat room temperature. ACU-PEG-Hydrazide was purified after centrifugeusing a spin column (1.8K MWCO). The product was lyophilized, andcharacterized by MALDI-TOF mass spectrometry. Finally, ACU-PEG-Hydrazide(12 mg) was reacted with N-4-acetylphenyl maleimide (1.2 mg) in DMFovernight at room temperature. The final product ACU-PEG-HZ-MAL waspurified through ether precipitation and obtained after lyophilization,which was characterized by MALDI-TOF mass spectrometry with a peakmolecular weight of 4,000 Da.

Preparation of ECO/pDNA and ACU-PEG-HZ-ECO/pDNA Nanoparticles

ECO (2.5 mM) in ethanol and ACU-PEG-HZ-MAL targeting ligand (0.4 mM inwater) (2.5 mol % of ECO) was first mixed and reacted in aqueoussolution for 30 min. Plasmid DNA (0.5 mg/mL) aqueous solution atpredetermined volume from the N/P ratio (amine to phosphate ratio) of 10were added to the ECO and ACU-PEG-HZ-MAL mixture, and vortexed for 30min at room temperature to give ACU-PEG-HZ-ECO/pDNA nanoparticles. AnECO stock solution (25 mM) of was used to formulate nanoparticles for invivo experiments. Encapsulations of pDNA by lipid ECO in nanoparticleformulations were characterized by an agarose gel electrophoresismethod. Agarose gel (0.7%) in TBE buffer was performed at 100 V for 30min.

Dynamic Light Scattering

The sizes and zeta potentials was characterized for nanoparticleformulations of ECO/pDNA (N/P=10), PEG-ECO/pDNA (N/P=10) andACU-PEG-HZ-ECO/pDNA (N/P=10) using a dynamic light scattering methodwith an Anton Paar Litesizer 500 (Anton Paar USA Inc, Ashland, Va.).Each sample was analyzed three times at 20° C.

In Vitro Transfection

Transfections were performed on 12-well plates, where ARPE-19 cells wereseeded at a concentration of 4×10⁴ cells/well. Cells were allowed togrow for 24 h before transfection. Nanoparticles at pDNA concentrationof 1 μg/mL in DMEM with 10% serum were added to ARPE-19 cells andincubated for 8 h at 37° C. The media containing nanoparticles was thenreplaced with fresh DMEM (10% serum). ARPE-19 cells were furtherincubated for an additional 48 h. Expression of ABCA4 was evaluated byqRT-PCR at mRNA level.

Transfection of ECO/pCMV-GFP nanoparticles of different N/P ratios werealso conducted similarly in ARPE-19 cells, where DMEM (10% serum) and apDNA concentration of 1 μg/mL were also used. The transfection wasperformed in 12-well plate with ARPE-19 cell concentration of 4×10⁴cells per well. The nanoparticles were formulated under N/P ratios of 6,8, 10 and 12 and were then incubated with ARPE-19 cells as previouslydescribed. After 24 h, fluorescence images of GFP expression wereacquired using an Olympus FV1000 confocal microscope.

Cytotoxicity

Cytotoxicity of ECO/pABCA4 and ACU4429 modified ECO/pABCA4 nanoparticleswas investigated using a CCK-8 assay (Dojindo Molecular Technologies,Inc, Washington, D.C.). Cell viability was evaluated using ARPE-19 cellson 96-well plates, where cells were seeded at a concentration of 1×10⁴cells per well. ARPE-19 cells were incubated with ECO/pABCA4 or ACU4429modified ECO/pABCA4 nanoparticles at DNA concentrations of 1 μg/mL inDMEM (10% serum) for 8 h at 37° C., after which, nanoparticle containingDMEM was replaced with fresh DMEM (10% serum). The cells were allowed togrow until 48 h and washed with PBS. The CCK-8 reagent was added to eachwell followed by an incubated of 1.5 h at 37° C. The absorbance at 450nm was recorded using a plate reader. Cell viability was characterizedby normalizing to the absorption of non-treated control. Free ECO, PEGligands and combinations were used as controls. The amount of eachmolecule is the same as what was used in each formulation.

qRT-PCR

For cells, a scraper was used for cell lysis and homogenization. Foranimal retinal tissues, eye samples were harvested from mice anddissected in a PBS buffer to separate neural retina and the RPE. Bothtissues were homogenized separately using a glass rod in a 1.5 mL tubeloaded with 0.6 mL of the lysis buffer. The RNA extractions for cellsand tissue samples were conducted using a QIAGEN RNeasy kit followingthe manufacturer's instructions. cDNAs were synthesized from mRNAtranscripts using a QIAGEN miScriptII reverse transcriptase kit(Germantown, MD). The qRT-PCR analysis was performed in a Mastercyclerinstrument (Eppendorf, Hauppauge, N.Y.) using a SYBR Green Master mix(AB Biosciences, Allston, Mass.). Fold changes of mRNA levels weredetermined by normalization to 18S. Eyes injected with PBS (SHAM) wereused as controls.

Intracellular Uptake

Intracellular uptake was first evaluated using microscopic method usingglass-bottom micro-well dishes, where ARPE-19 cells were seeded at aconcentration of 4×10⁴ cells/dish and grew for 24 h at 37° C. Then, thecell nuclei were stained with Hoechst 33342 (4 μg/mL) (Invitrogen) andendosomes/lysosomes with LysoTracker Green (100 mM) (Life Technologies,Carlsbad, Calif.). ARPE-19 cells were then incubated withACU-PEG-HZ-ECO/Cy3-pDNA or PEG-ECO/Cy3-pDNA (N/P=10) nanoparticles inDMEM (10% serum) for 1 h, 2 h, 4 h and 24 h (where nanoparticlecontaining media was replaced by fresh DMEM at 4 h and cells werefurther incubated for 24 h). After incubation, cells were washed withPBS three times and then fixed in 0.5 mL of PBS containing 4%paraformaldehyde (4% PFA). Samples were imaged for cy3 fluorescenceusing an Olympus FV1000 confocal microscope (Olympus, Center Valley,Pa.). Quantitative analysis was performed using a flow cytometry method.To prepare samples, cells were washed with PBS and then harvested aftertreatment with trypsin (0.25%, 0.26 mmol EDTA) (Invitrogen, Carlsbad,Calif.). The cell pellets were obtained by centrifugation (1500 rpm, 5min) and resuspended in 4% PFA (0.5 mL). The cell suspensions werefinally forced through a cell strainer (35 μm) (BD Biosciences, FranklinLakes, N.J.). Cy3 positive ARPE-19 cells were quantified by measurementsof the fluorescence intensities for more than 3,000 cells from eachsample using a BD FACSCalibur flow cytometer (BD Biosciences, FranklinLakes, N.J.).

In Vivo Subretinal Transfection with PEG-ECO/pDNA andACU-PEG-HZ-ECO/pDNA Nanoparticles

Subretinal injection was performed as previously described. Thenanoparticle solution (1 μL) was injected by a pump with a steady speedof 150 nL/sec into the mouse eye. Successful administration wasconfirmed by bleb formation in the subretinal space. A total of 200 ngof Cy3-labeled plasmid or pABCA4 (CMV or RHO promoters) was delivered.The contralateral eye injected with 1 μL of PBS was used as control. Atleast three days after the subretinal administration, examinations usingOCT were performed to ensure the heal of retinal structure of theinjection site with minimal retinal detachment.

Immunohistochemistry

Histological samples were prepared as previously described (14). Theeyes were harvested from mice after subretinal injections, washed withPBS, and fixed in 4% PFA for 2 h. Then eye cups were prepared andfurther fixed in 4% PFA overnight. The eyecups were then immersed inTissue-Tek optimal cutting temperature compound (OCT) (Sakura, Torrance,Calif.) solutions containing 20% sucrose through a gradual sucrosegradient. After incubation in 20% sucrose/OCT overnight, the eye cupswere imbedded in cryomolds and flash frozen in OCT. Histological slides(10 μm-thick) were collected under frozen conditions. The slides werethen warmed to room temperature and further washed with Tris buffercontaining 1% tween 20 (TBST) buffer. Followed by an antigen retrieval(20 min) in citrate buffer (10 mM, pH 6.0) using a pressure cooker, theslides were ready for staining. DAPI was used to label cell nuclei.Fluorescence images of the distribution of Cy3-labeled pDNA and DAPIwere collected using an Olympus FV1000 confocal microscope (Olympus).

Statistical Analysis

Experiments were performed in triplicate and the number of animals islisted in the figure captions. Experimental data are presented asaverages with standard deviations. Statistical analysis was performedwith one-way ANOVA and two-tailed Student's t-tests. A 95% confidenceinterval was used and P<0.05 was accepted as statistically significant.

Results

The ACU4429 targeting ligand with a PEG spacer and a hydrazone linker(ACU-PEG-HZ-MAL) was synthesized as described in FIG. 8A. An excess ofACU4429 was first reacted with a 3.4 kD HS-PEG-NHS to give ACU-PEG-SH. Ahydrazide group was then introduced to the ACU-PEG-SH by reacting withan excess of EMCH to form ACU-PEG-CO—NHNH₂, which then reacted withN-4-acetylphenyl maleimide to give the targeting ligand with a PEGspacer and a HZ linker ACU4429-PEG-HZ-MAL. The maleimido group wasintroduced for conjugating to one of the thiol groups on ECO. Theintermediates and ACU-PEG-HZ-MAL were purified by precipitation andcharacterized by MALDI-TOF mass spectrometry (FIG. 8B). The molecularpeak shifts in the spectra toward higher molecular weight indicatedformation of desired products during the synthesis of ACU-PEG-HZ-MAL ateach step.

To formulate targeted ECO/pABCA4 nanoparticles, ACU-PEG-HZ-MAL targetingligand (2.5 mol-%) was first mixed and reacted with ECO molecules inaqueous solution for 30 min. The targeted nanoparticles then formed byself-assembly when pABCA4 was added to the solution (FIG. 9A).ECO/pABCA4 nanoparticles (no ligand) and PEG-ECO/pABCA4 (non-targetingligand 3.4 kD PEG chain conjugated) nanoparticles were similarlyprepared as controls. The nanoparticles were characterized using dynamiclight scattering. All nanoparticles had similar sizes and sizedistribution with a diameter of 195.91±12.93 nm for ECO/pABCA4,199.54±0.74 nm for PEG-ECO/pABCA4 and 195.13±5.74 nm forACU-PEG-HZ-ECO/pABCA4 (FIG. 9B,C). DLS zeta potential measurementsshowed that the nanoparticles had positive surface charge, with39.05±0.64 mV for ECO/pABCA4 and 33.51±0.71 mV for PEG-ECO/pABCA4 and33.83±0.40 mV for ACU-PEG-HZ-ECO/pABCA4 nanoparticles (FIG. 9D,E). Todetermine the best N/P (amine/phosphate) ratio for effectiveintracellular gene delivery, transfections in ARPE-19 cells wereperformed using ECO/pCMV-GFP nanoparticles formulated at N/P ratio 6, 8,10 and 12. Confocal images of GFP expression (FIG. 9F) demonstratedincreased GFP expression with N/P ratios. However, potential increasedcytotoxicity was observed when the N/P ratio was 12. We selected N/Pratio of 10 for the following evaluations, due to a potential highertransfection efficiency of delivering large genes.

To evaluate the stability and encapsulation of ECO/pABCA4 and ACU4429modified ECO/pABCA4 nanoparticles, an agarose gel electrophoresis wasperformed. The complete encapsulation of pABCA4 (16 kb) was observed inthe nanoparticles with no free DNA smear in the gels using the standardnanoparticle formulation procedure (FIG. 10A). The bands on the top ofthe gel of ECO/pABCA4 and modified ECO/pABCA4 nanoparticles demonstratedlimited mobility of encapsulated pABCA4, indicating good stability ofthese nanoparticles. However, if ECO and pABCA4 were mixed shortly onlyby pipetting, DNA was not completely encapsulated and a free pABCA4smear was observed (black arrow). Controls of free ECO and PEG ligandsdid not show any bands in the gel. The introduction of the ACU-4429targeting ligand with a PEG spacer produced only slight reduction ofzeta potential that did not affect the size and encapsulation ofECO/pABCA4 nanoparticles. To evaluate cytotoxicity of ECO/pABCA4 andACU4429 modified ECO/pABCA4 nanoparticles, a CCK-8 assay was performedin ARPE-19 cells (FIG. 10B). No significant cytotoxicity was observedfor ECO/pABCA4 and modified ECO/pABCA4 nanoparticles compared with thenon-treated control. Free ECO, PEG ligands and combinations of both alsodid not demonstrated significant cytotoxicity (p<0.05).

To evaluate the intracellular transfection efficiency ofACU-PEG-HZ-ECO/pDNA nanoparticles, ARPE-19 cells were transfected withACU-PEG-HZ-ECO/Cy3-pDNA nanoparticles at N/P=10, with the non-targetedPEG-ECO/Cy3-pDNA nanoparticles as a control. Fluorescence confocalmicroscopy clearly revealed the differences in intracellular uptake anddistribution of the fluorescently labeled DNA between these nanoparticleformulations. The ACU4429 targeted nanoparticles with the hydrazonelinker demonstrated stronger cellular uptake at an early time point witha dispersed cytoplasmic DNA distribution at later time points (FIG.11A). At 1 h, both nanoparticles interacted with the cell membrane andbegan internalization. At 2 h, both nanoparticles were internalized bythe cells. It seems that the targeted nanoparticles had higher cellularuptake than the non-targeted nanoparticles. At later time points,especially at 24 h, a highly dispersed distribution of the labeled DNAwas observed in the cytoplasm of the cells treated withACU-PEG-HZ-ECO/Cy3-pDNA nanoparticles, while only spotted intracellulardistribution was observed with PEG-ECO/Cy3-pDNA nanoparticles (FIG.11A). The intracellular transfections of both nanoparticle systems werealso quantified using a flow cytometry analysis (FIG. 11B), which wasconsistent with the confocal observations. ACU-PEG-HZ-ECO/Cy3-pDNAnanoparticles demonstrated higher mean fluorescence intensities for allthe time points. These results suggest excellent endosomal escapeability of ACU-PEG-HZ-ECO/Cy3-pDNA nanoparticles. The excellentendosomal escape ability was translated to high gene expressionefficiency in the ARPE-19 cells transfected by ACU-PEG-HZ-ECO/pABCA4nanoparticles (FIG. 11C). Significantly higher ABCA4 mRNA expression wasobserved for ACU-PEG-HZ-ECO/pABCA4 nanoparticles than PEG-ECO/pABCA4nanoparticles 48 h after transfection. Taken together, the targetedsmart ACU-PEG-HZ-ECO/pDNA nanoparticles improved endosomal escape andcytosolic DNA delivery efficiency, and resulted in higher levels of geneexpression.

To evaluate the in vivo targeting efficiency, ACU-PEG-HZ-ECO/Cy3-pDNAnanoparticles were injected into the subretinal space in Abca4^(−/−)mice. ACU-PEG-HZ-ECO/Cy3-pDNA demonstrated excellent targetingefficiency in the subretinal space, where abundant IRBP is expressed(FIG. 12A). ACU-PEG-HZ-ECO/Cy3-pDNA nanoparticles remained in theinterphotoreceptor matrix (IPM), demonstrated by the red signals in thesubretinal space. In comparison, the non-targeted ECO/Cy3-pDNAnanoparticles diffused in both the RPE and photoreceptors.

Binding of ACU-PEG-HZ-ECO/pDNA nanoparticles to IRBP in the IPM resultedin enhanced RPE specific gene expression after subretinal injections ofthe nanoparticles. Both ACU-PEG-HZ-ECO/pABCA4 and PEG-ECO/pABCA4nanoparticles were formulated with a photoreceptor-specific RHO promoteror a common CMV promoter by self-assembly. The nanoparticles (1 μL) wereinjected into the subretinal space of Abca4^(−/−) mice at a dose of 200ng DNA. For non-targeted ECO/pCMV-ABCA4 nanoparticles, similar ABCA4mRNA expression in the retina and the RPE with the common promoter wasdemonstrated, while significantly higher ABCA4 mRNA expression in theretina was achieved with PEG-ECO/pRHO-ABCA4 by using the specific RHOpromoter (FIG. 12B). For the ACU4429 targeted nanoparticles, more ABCA4mRNA expression was observed in the RPE than the retina forACU-PEG-HZ-ECO/pCMV-ABCA4 because of enhanced RPE uptake mediated byACU4429. Due to the photoreceptor specificity of the RHO promoter inconjunction with the targeting effect of ACU-4429 to the RPE and lowgene expression with pRHO-ABCA4 in the RPE cells, no statisticaldifference between ABCA4 mRNA expression in the retina and the RPE wasfound with ACU-PEG-HZ-ECO/pRHO-ABCA4. Taken together, the resultsindicated that the stable retinylamine analogue ACU4429 modifiedECO/pDNA nanoparticles effectively facilitated delivery of therapeuticDNA and induced gene expression in the RPE.

We have shown here that modification of ECO/pDNA nanoparticles withACU4429, a stable all-trans-retinoid analogue, can also enhance genedelivery to the RPE through binding to IRBPs. It is speculated thatafter injection of the ACU-PEG-HZ-ECO/pDNA nanoparticles into thesubretinal space, ACU4429 would quickly bind to IRBPs, which would thentransport the nanoparticles near the apical surface of the RPE andfacilitate the internalization of the nanoparticles by the RPE (FIG.13). Consequently, more Cy3-pDNA and expression of pABCA4 plasmid wasobserved in the RPE than the retina when the ECO/pDNA nanoparticles weremodified with ACU4429 as a targeting ligand.

PEGylation is a common strategy in the design of target gene deliveryplatforms to minimize non-specific cellular uptake and potential toxicside effects. However, PEGylation compromises the efficiency ofendosomal escape and cytosolic release of the gene cargo. In order toovercome this obstacle, a pH-sensitive hydrazone linker was used toconjugate the targeting agent with the PEG spacer to shed the PEG layerthrough a pH-sensitive hydrolysis of hydrazone once targetednanoparticles were in acidic endosomes. The core ECO/pDNA nanoparticleswould be exposed to enhance endosomal escape and cytosolic release ofthe DNA cargo through the PERC effect of ECO. The enhancedamphiphilicity of ECO due to the protonation of the amino head groups ofECO in the acidic endosomes (pH=5-6) would destabilize the endosomalmembrane to facilitate the escape of the ECO/pDNA nanoparticles intocytosol. The reductive environment of the cytosol would reduce thedisulfide bonds in the nanoparticles, lead to dissociation of theECO/pDNA formulations and release the DNA payloads. In contrast, theregular PEGylated ECO/pDNA nanoparticles had limited ability to escapefrom the endosomal entrapment. The results in this work have shown thatthe strategy of using a pH-sensitive hydrazone linker facilitatedendosomal escape and cytosolic release of the DNA payload of thenanoparticles and significantly enhanced the gene expression of theACU-PEG-HZ-ECO/pDNA as compared to PEG-ECO/pDNA nanoparticles.

Recently, ABCA4 expression was detected in the mouse RPE, comprising 1%of its level in photoreceptors of the neural retina. Expression of ABCA4in the RPE has a partial response in the ocular Abca4^(−/−) phenotype.Delivery and expression of ABCA4 gene in the RPE cells have thepotential to prevent the progression of Stargardt disease. We used ABCA4plasmids with a common CMV promoter and a rod photoreceptor specific RHOpromoter to demonstrate the ability of ACU4429 targeted ECO/pDNAnanoparticles for specific delivery of a large ABCA4 gene in the RPE.More significant expression of ABCA4 was observed for the targetednanoparticles with the CMV promoter in the RPE than in the retina, whilethe targeted nanoparticles with the RHO promoter had lower relativeABCA4 expression in the retina than the non-targeted nanoparticles. Theresults have validated the hypothesis that the targeted ECO/pDNAnanoparticles are effective for specific delivery of therapeutic genesinto the RPE in vivo. ACU4429 modified ECO/pDNA nanoparticles have thepromise to be used as non-viral system to deliver therapeutic genes ofany sizes to the RPE for gene therapy of monogenetic visual disorders.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, we claim:
 1. A nanosized complexcomprising: a nucleic acid that is complexed with a compound, thecompound including formula (I):

wherein R¹ is an alkylamino group or a group containing at least onearomatic group; R² and R³ are independently an aliphatic group or ahydrophobic group; R⁴ and R⁵ are independently H, a substituted orunsubstituted alkyl group, an alkenyl group, an acyl group, or anaromatic group, or a polymer, a targeting group, a detectable moiety, ora linker, or a combination thereof, and at least one of R⁴ and R⁵includes a retinoid or retinoid derivative that targets and/or binds toan interphotoreceptor retinoid binding protein; a, b, c, and d areindependently an integer from 1 to 10; and pharmaceutically acceptablesalts thereof.
 2. The complex of claim 1, wherein R¹ comprises at leastone of:

where R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are independentlyhydrogen, an alkyl group, a hydrophobic group, or a nitrogen containingsubstituent; and e, f, g, i, j, k, and 1, are an integer from 1 to 10.3. The complex of claim 1, wherein R² and R³ are independently ahydrophobic group derived from oleic acid or linoleic acid.
 4. Thecomplex of claim 3, wherein R² and R³ are the same.
 5. The complex ofclaim 1, wherein at least one of R⁴ or R⁵ includesall-trans-retinylamine or(1R)-3-amino-1-[3-(cyclohexylmethoxy)phenyl]propan-1-ol.
 6. The complexof claim 1, wherein a, b, c, and d are each
 2. 7. The complex of claim1, R¹ comprises at least one of CH₂CH₂NH₂, CH₂CH₂NHCH₂CH₂NHCH₂CH₂NH, orCH₂CH₂NHCH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NH.
 8. The complex of claim 1, whereinthe retinoid or retinoid derivative is covalently attached to a thiolgroup of a cysteine residue by a linker.
 9. The complex of claim 8,wherein the linker comprises a polyamino acid group, a polyalkylenegroup, or a polyethylene glycol group.
 10. The complex of claim 8,wherein the linker comprises an acid labile bond.
 11. The complex ofclaim 1, wherein the nucleic acid is capable of treating, ameliorating,attenuating, and/or eliminating symptoms of a disease or disorder of theeye when the complex is introduced to or within the eye of a subjectwith a disease or disorder.
 12. The complex of claim 11, wherein thenucleic acid comprises a siRNA or plasmid DNA.
 13. The complex of claim10, wherein the nucleic acid includes plasmid DNA encoding at least oneof an ATP-binding cassette transporter, RPE65, RHO, RdCVF, or CP290. 14.A method of treating a retinal disorder in a subject in need thereof,the method comprising: administering to the subject a nanosized complexcomprising: a nucleic acid that is complexed with a compound, whereinthe nucleic acid is capable of treating, ameliorating, attenuating,and/or eliminating symptoms of the retinal disorder when the complex isintroduced to or within an eye of a subject, and wherein the compoundincludes formula (I):

wherein R¹ is an alkylamino group or a group containing at least onearomatic group; R² and R³ are independently an aliphatic group or ahydrophobic group; R⁴ and R⁵ are independently H, a substituted orunsubstituted alkyl group, an alkenyl group, an acyl group, or anaromatic group, or a polymer, a targeting group, a detectable moiety, ora linker, or a combination thereof, and at least one of R⁴ and R⁵includes a retinoid or retinoid derivative that targets and/or binds toan interphotoreceptor retinoid binding protein; a, b, c, and d areindependently an integer from 1 to 10; and pharmaceutically acceptablesalts thereof.
 15. The method of claim 14, wherein R² and R³ areindependently a hydrophobic group derived from oleic acid or linoleicacid.
 16. The method of claim 14, wherein at least one of R⁴ or R⁵includes all-trans-retinylamine or(1R)-3-amino-1-[3-(cyclohexylmethoxy)phenyl]propan-1-ol.
 17. The methodof claim 14, R¹ comprises at least one of CH₂CH₂NH₂,CH₂CH₂NHCH₂CH₂NHCH₂CH₂NH, or CH₂CH₂NHCH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NH.
 18. Themethod of claim 14, wherein the retinoid or retinoid derivative iscovalently attached to a thiol group of a cysteine residue by a linker.19. The method of claim 18, wherein the linker comprises a polyaminoacid group, a polyalkylene group, or a polyethylene glycol group. 20.The method of claim 19, wherein the linker comprises an acid labilebond.
 21. The method of claim 14, wherein the retinal disorder comprisesat least one of Leber congenital amaurosis, Stargardt disease, orretinitis pigmentosis and the nucleic acid includes a plasmid DNAencoding at least one of an ATP-binding cassette transporter, RPE65, orRHO.